bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme Characteristics of β-
d
-Galactosidase- and β-
d
-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and
Escherichia coli
Authors: Tryland I. , Fiksdal L.
Published: December 2019

Bacteria which were β-
d
-galactosidase and β-
d
-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-
d
-galactoside and 4-methylumbelliferyl-β-
d
-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and
Escherichia coli
were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-
d
-galactosidase-positive nontarget bacteria but none of the β-
d
-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-
d
-galactosidase- and β-
d
-glucuronidase-positive bacteria. The induced and noninduced β-
d
-glucuronidase activities of
Bacillus
spp. and
Aerococcus viridans
were approximately the same as the activities of induced
E. coli
. Except for some isolates identified as
Aeromonas
spp., all of the induced and noninduced β-
d
-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-
d
-galactosidase activity than induced
E. coli
. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.
Detection of
Escherichia coli
O157:H7 in the Beef Marketed in Malaysia
Authors: Radu Son , Mutalib Shahilah Abdul , Rusul Gulam , Ahmad Zainori , Morigaki Tadaaki , Asai Norio , Kim Yung Bu , Okuda Jun , Nishibuchi Mitsuaki
Published: December 2019

Twelve strains of
Escherichia coli
O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the
eae
gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.
Direct Detection of Viable Bacteria, Molds, and Yeasts by Reverse Transcriptase PCR in Contaminated Milk Samples after Heat Treatment
Authors: Vaitilingom Marc , Gendre Francois , Brignon Pierre
Published: December 2019

A fast, sensitive, and target contaminant-modulable method was developed to detect viable bacteria, molds, and yeasts after heat treatment. By reverse transcriptase PCR with elongation factor gene (EF-Tu or EF-1α)-specific primers, the detection level was 10 cells ml of milk
−1
. The simplicity and rapidity (4 h) of the procedure suggests that this method may be easily transposable to other foods and other contaminants.
2-Chloro-1,4-Dimethoxybenzene as a Novel Catalytic Cofactor for Oxidation of Anisyl Alcohol by Lignin Peroxidase
Authors: Teunissen Pauline J. M. , Field Jim A.
Published: December 2019

2-Chloro-1,4-dimethoxybenzene (2Cl-14DMB) is a natural compound produced de novo by several white rot fungi. This chloroaromatic metabolite was identified as a cofactor superior to veratryl alcohol (VA) in the oxidation of anisyl alcohol (AA) by lignin peroxidase (LiP). Our results reveal that good LiP substrates, such as VA and tryptophan, are comparatively poor cofactors in the oxidation of AA. Furthermore, we show that a good cofactor does not necessarily serve a role in protecting LiP against H
2
O
2
inactivation. 2Cl-14DMB was not a direct mediator of AA oxidation, since increasing AA concentrations did not inhibit the oxidation of 2Cl-14DMB at all. However, the high molar ratio of anisaldehyde formed to 2Cl-14DMB consumed, up to 13:1, indicates that a mechanism which recycles the cofactor is present.
Isolation, Characterization, and Transfer of Cryptic Gene-Mobilizing Plasmids in the Wheat Rhizosphere
Authors: van Elsas Jan Dirk , McSpadden Gardener Brian B. , Wolters Anneke C. , Smit Eric
Published: December 2019

A set of self-transmissible plasmids with IncQ plasmid-mobilizing capacity was isolated by triparental exogenous isolation from the wheat rhizosphere with an
Escherichia coli
IncQ plasmid host and a
Ralstonia eutropha
recipient. Three plasmids of 38 to 45 kb, denoted pIPO1, pIPO2, and pIPO3, were selected for further study. No selectable traits (antibiotic or heavy-metal resistance) were identified in these plasmids. The plasmids were characterized by replicon typing via PCR and hybridization with replicon-specific probes and other hybridizations. pIPO1 and pIPO3 were similar to each other, whereas pIPO2 was different. None of these plasmids belonged to any known incompatibility group. pIPO2 was selected for further work, and a mini-Tn
5-tet
transposon was inserted to confer selectability. Plasmid pIPO2 had a broad IncQ plasmid mobilization and self-transfer range among the alpha, beta, and gamma subclasses of the
Proteobacteria
but did not show productive transfer to gram-positive bacteria. Plasmid pIPO2 mobilized IncQ plasmid pIE723 from
Pseudomonas fluorescens
to diverse indigenous proteobacteria in the rhizosphere of field-grown wheat. Transfer of pIE723 to indigenous bacteria was not observed in the absence of added pIPO2. A specific PCR primer system and a probe were developed for the detection of pIPO2-type plasmids in soil and rhizosphere. Analysis of soil DNA provided evidence for the presence of pIPO2 in inoculated wheat rhizosphere soil in the field study, as well as in the rhizosphere of uninoculated wheat plants growing in soil microcosms. The system failed to identify major reservoirs of pIPO2 in a variety of other soils.
Detection of
Clostridium proteoclasticum
and Closely Related Strains in the Rumen by Competitive PCR
Authors: Reilly K. , Attwood G. T.
Published: December 2019

A competitive PCR technique was used to enumerate the proteolytic bacterium
Clostridium proteoclasticum
from the rumen. A PCR primer, which circumscribes this organism and several closely related strains, was designed for a variable region within their 16S rRNA genes and was used in conjunction with a universal forward primer. This primer pair was tested for specificity against 85 ruminal bacterial strains. An internal control DNA was constructed for use in competitive PCRs and was shown to amplify under the same reaction conditions and with the same amplification efficiency as the target DNA. DNA from a known number of
C. proteoclasticum
cells was coamplified with the internal control to construct a standard curve. Rumen samples were collected from eight dairy cows fed four diets in rotation: high nitrogen, high nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen supplemented with carbohydrate. DNA extracted from these and spiked with internal control DNA was amplified with the
C. proteoclasticum
primer pair. The relative intensities of the PCR products were used to quantitate the numbers of
C. proteoclasticum
cell equivalents from the rumen samples. The numbers ranged from 2.01 × 10
6
ml
−1
to 3.12 × 10
7
ml
−1
. There was no significant effect on the numbers of
C. proteoclasticum
detected in rumen samples among cows fed the four diets. The utility of the competitive PCR approach for quantifying ruminal bacterial populations in vivo and the occurrence of
C. proteoclasticum
in forage-fed dairy cows are discussed.
Comparative Recoveries of
Naegleria fowleri
Amoebae from Seeded River Water by Filtration and Centrifugation
Authors: Pernin P. , Pélandakis M. , Rouby Y. , Faure A. , Siclet F.
Published: December 2019

Detection of pathogenic
Naegleria fowleri
in environmental water samples, which is necessary for the prevention of primary amoebic meningoencephalitis, generally requires concentrating the samples. Two concentration techniques, filtration and centrifugation, were used to study the recovery of
N. fowleri
, in vegetative or cystic form, that had been mixed with the two other thermotolerant
Naegleria
species,
N. lovaniensis
and
N. australiensis
. Counting of amoebae was performed by the most probable number method on 10 water replicates of 100 ml and 10 ml each. With both concentration methods, recovery was better for cysts than for trophozoites (53% ± 21% versus 5% ± 5% by filtration and 57% ± 25% versus 22% ± 5% by centrifugation). The recovery of
Naegleria
trophozoites by filtration was very low, and centrifugation was significantly better than filtration in recovery of
Naegleria
trophozoites (22% ± 5% versus 5% ± 5%;
P
< 0.001). For cysts, however, filtration appeared as efficient as centrifugation, with equivalent values for recovery (53% ± 21% versus 57% ± 25%;
P
> 0.7). Although the recovery of cysts of
N. fowleri
obtained by filtration (51% ± 24%) appeared higher than that by centrifugation (36% ± 23%), the difference was not significant (
P
> 0.1). Both concentration methods have highly variable recovery rates, making accurate quantification of low concentrations (<100/liter) of
N. fowleri
in the environment difficult.
Alkane Hydroxylase from
Acinetobacter
sp. Strain ADP1 Is Encoded by
alkM
and Belongs to a New Family of Bacterial Integral-Membrane Hydrocarbon Hydroxylases
Authors: Ratajczak Andreas , Geißdörfer Walter , Hillen Wolfgang
Published: December 2019

Degradation of long-chain alkanes by
Acinetobacter
sp. strain ADP1 involves rubredoxin and rubredoxin reductase. We complemented a mutant deficient in alkane utilization and sequenced four open reading frames (ORFs) on the complementing DNA. Each of these ORFs was disrupted by insertional mutagenesis on the chromosome. As determined from sequence comparisons, ORF1 and ORF4 seem to encode a rotamase of the PpiC type and an acyl coenzyme A dehydrogenase, respectively. Disruption of these ORFs does not affect alkane utilization. In contrast, the two other ORFs,
alkR
and
alkM
, are essential for growth on alkanes as sole carbon sources.
alkR
encodes a polypeptide with extensive homology to AraC-XylS-like transcriptional regulators. It is located next to
alkM
, which encodes the terminal alkane hydroxylase, but is in the opposite orientation. Sequence homologies with other bacterial integral-membrane hydrocarbon hydroxylases suggest that AlkM may be the first member of a new protein family. The genes identified here are not linked to the rubredoxin- and rubredoxin reductase-encoding genes on the
Acinetobacter
sp. strain ADP1 chromosome.
Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of
Pseudomonas syringae
Authors: Clerc Agathe , Manceau Charles , Nesme Xavier
Published: December 2019

Recently, DNA pairing analyses showed that
Pseudomonas syringae
pv. tomato and related pathovars, including
P. syringae
pv. maculicola, form a genomic species (
Pseudomonas tomato
) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445–448,
in
K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed.,
Pseudomonas syringae Pathovars and Related Pathogens
, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of
P. syringae
cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of
P. syringae
strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of
P. syringae
pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other
P. syringae
pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of
P. syringae
pv. tomato and
P. syringae
pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of
P. syringae
pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.
Phase Variation in
Xenorhabdus nematophilus
Authors: Volgyi Antonia , Fodor Andras , Szentirmai Attila , Forst Steven
Published: December 2019

Xenorhabdus nematophilus
is a symbiotic bacterium that inhabits the intestine of entomopathogenic nematodes. The bacterium-nematode symbiotic pair is pathogenic for larval-stage insects. The phase I cell type is the form of the bacterium normally associated with the nematode. A variant cell type, referred to as phase II, can form spontaneously under stationary-phase conditions. Phase II cells do not elaborate products normally associated with the phase I cell type. To better define phase variation in
X. nematophilus
, several strains (19061, AN6, F1, N2-4) of this bacterium were analyzed for new phenotypic traits. An analysis of pathogenicity in
Manduca sexta
larvae revealed that the phase II form of AN6 (AN6/II) was significantly less virulent than the phase I form (AN6/I). The variant form of N2-4 was also avirulent. On the other hand, F1/II and 19061/II were as virulent as the respective phase I cells. Strain 19061/II was found to be motile, and AN6/II regained motility when the bacteria were grown in low-osmolarity medium. In contrast, F1/II remained nonmotile. The phase II cells did not produce the outer membrane protein, OpnB, that is normally induced during the stationary phase. Both phase I and phase II cells were able to support nematode growth and development. These findings indicate that while certain phenotypic traits are common to all phase II cells, other characteristics, such as virulence and motility, are variable and can be influenced by environmental conditions.
Analysis of BIOLOG GN Substrate Utilization Patterns by Microbial Communities
Authors: Smalla Kornelia , Wachtendorf Ute , Heuer Holger , Liu Wen-tso , Forney Larry
Published: December 2019

BIOLOG GN plates are increasingly used to characterize microbial communities by determining the ability of the communities to oxidize various carbon sources. Studies were done to determine whether the BIOLOG GN plate assay accurately reflects the catabolic potential of the inoculum used. To gain insight into which populations of microbial communities contribute to the BIOLOG patterns, denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis (TGGE) were used to assess the diversity of ribotypes in the inocula and individual wells of BIOLOG plates following incubation. These studies were done with microbial communities from the rhizosphere of potatoes and an activated sludge reactor fed with glucose and peptone. TGGE analyses of BIOLOG wells inoculated with cell suspensions from the potato rhizosphere revealed that, compared with the inoculum, there was a decrease in the number of 16S rRNA gene fragments obtained from various wells, as well as a concomitant loss of populations that had been numerically dominant in the inoculum. The dominant fragments in TGGE gels could be assigned to the γ subclass of the class
Proteobacteria
, suggesting that fast-growing bacteria adapted to high substrate concentrations were numerically dominant in the wells and may have been primarily responsible for the patterns of substrate use that were observed. Similarly, the community structure changed in wells inoculated with cells from activated sludge; one or more populations were enriched, but all dominant populations of the inoculum could be detected in at least one well. This study showed that carbon source utilization profiles obtained with BIOLOG GN plates do not necessarily reflect the functional potential of the numerically dominant members of the microbial community used as the inoculum.
Development of a Laboratory-Scale Leaching Plant for Metal Extraction from Fly Ash by
Thiobacillus
Strains
Authors: Brombacher Christoph , Bachofen Reinhard , Brandl Helmut
Published: December 2019

Semicontinuous biohydrometallurgical processing of fly ash from municipal waste incineration was performed in a laboratory-scale leaching plant (LSLP) by using a mixed culture of
Thiobacillus thiooxidans
and
Thiobacillus ferrooxidans
. The LSLP consisted of three serially connected reaction vessels, reservoirs for a fly ash suspension and a bacterial stock culture, and a vacuum filter unit. The LSLP was operated with an ash concentration of 50 g liter
−1
, and the mean residence time was 6 days (2 days in each reaction vessel). The leaching efficiencies (expressed as percentages of the amounts applied) obtained for the economically most interesting metal, Zn, were up to 81%, and the leaching efficiencies for Al were up to 52%. Highly toxic Cd was completely solubilized (100%), and the leaching efficiencies for Cu, Ni, and Cr were 89, 64, and 12%, respectively. The role of
T. ferrooxidans
in metal mobilization was examined in a series of shake flask experiments. The release of copper present in the fly ash as chalcocite (Cu
2
S) or cuprite (Cu
2
O) was dependent on the metabolic activity of
T. ferrooxidans
, whereas other metals, such as Al, Cd, Cr, Ni, and Zn, were solubilized by biotically formed sulfuric acid. Chemical leaching with 5 N H
2
SO
4
resulted in significantly increased solubilization only for Zn. The LSLP developed in this study is a promising first step toward a pilot plant with a high capacity to detoxify fly ash for reuse for construction purposes and economical recovery of valuable metals.
19
F Nuclear Magnetic Resonance as a Tool To Investigate Microbial Degradation of Fluorophenols to Fluorocatechols and Fluoromuconates
Authors: Boersma Marelle G. , Dinarieva Tatiana Y. , Middelhoven Wouter J. , van Berkel Willem J. H. , Doran Joel , Vervoort Jacques , Rietjens Ivonne M. C. M.
Published: December 2019

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by
19
F nuclear magnetic resonance (NMR). Characterization of the
19
F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus
Exophiala jeanselmei
, provided possibilities for identification of the
19
F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the
19
F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of
E. jeanselmei
. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a
19
F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.
Reductive Dechlorination of Tetrachloroethene to Ethene by a Two-Component Enzyme Pathway
Authors: Magnuson Jon K. , Stern Robert V. , Gossett James M. , Zinder Stephen H. , Burris David R.
Published: December 2019

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing
Dehalococcoides ethenogenes
195. When titanium(III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 μmol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE,
cis
-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 μmol/min/mg of protein. Vinyl chloride and
trans
-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.
Oxygen-Dependent Regulation of the Expression of the Catalase Gene
katA
of
Lactobacillus sakei
LTH677
Authors: Hertel Christian , Schmidt Gudrun , Fischer Marc , Oellers Katja , Hammes Walter P.
Published: December 2019

The catalase gene
katA
of
Lactobacillus sakei
LTH677 was cloned and expressed in
Escherichia coli
UM2,
Lactobacillus casei
LK1, and
Lactobacillus curvatus
LTH1432. The last host is a catalase-deficient plasmid-cured derivative of a starter organism used in meat fermentation. The regulation of
katA
expression was found to be the same in
L. sakei
LTH677 and the recombinant strains. The addition of H
2
O
2
to anaerobic cultures, as well as a switch to aerobic conditions, resulted in a strong increase in KatA activity. The expression was investigated in more detail with
L. sakei
LTH677 and
L. curvatus
LTH4002. The recombinant strain LTH4002 did not accumulate H
2
O
2
under glucose-limited aerobic conditions and remained viable in the stationary phase. Under inductive conditions, the
katA
-specific mRNA and the apoenzyme were synthesized de novo. Deletion derivatives of the
katA
promoter were produced, and the regulatory response was investigated by fusion to the β-glucuronidase reporter gene
gusA
and expression in
L. sakei
LTH677. The fact that gene expression was subject to induction was confirmed at the level of transcription and protein synthesis. A small putative regulatory sequence of at least 25 bp was identified located upstream of the −35 site. Competition experiments performed with
L. sakei
LTH677 harboring the fusion constructs consisting of the
katA
promoter and
gusA
revealed that an activator protein is involved in the transcriptional induction of
katA.
Differential Effects of Dimethylsulfoniopropionate, Dimethylsulfonioacetate, and Other S-Methylated Compounds on the Growth of
Sinorhizobium meliloti
at Low and High Osmolarities
Authors: Pichereau Vianney , Pocard Jean-Alain , Hamelin Jack , Blanco Carlos , Bernard Théophile
Published: December 2019

An extract from the marine alga
Ulva lactuca
was highly osmoprotective in salt-stressed cultures of
Sinorhizobium meliloti
102F34. This beneficial activity was due to algal 3-dimethylsulfoniopropionate (DMSP), which was accumulated as a dominant compatible solute and strongly reduced the accumulation of endogenous osmolytes in stressed cells. Synthetic DMSP also acted as a powerful osmoprotectant and was accumulated as a nonmetabolizable cytosolic osmolyte (up to a concentration of 1,400 nmol/mg of protein) throughout the growth cycles of the stressed cultures. In contrast, 2-dimethylsulfonioacetate (DMSA), the sulfonium analog of the universal osmoprotectant glycine betaine (GB), was highly toxic to unstressed cells and was not osmoprotective in stressed cells of wild-type strains of
S. meliloti
. Nonetheless, the transport and accumulation of DMSA, like the transport and accumulation of DMSP and GB, were osmoregulated and increased fourfold in stressed cells of strain 102F34. Strikingly, DMSA was not toxic and became highly osmoprotective in mutants that are impaired in their ability to demethylate GB and DMSA. Furthermore, 2-methylthioacetate and thioglycolic acid (TGA), the demethylation products of DMSA, were excreted, apparently as a mechanism of cellular detoxification. Also, exogenous TGA and DMSA displayed similar inhibitory effects in strain 102F34. Thus, on the basis of these findings and other physiological and biochemical evidence, we infer that the toxicity of DMSA in wild-type strains of
S. meliloti
stems from its catabolism via the GB demethylation pathway. This is the first report describing the toxicity of DMSA in any organism and a metabolically stable osmoprotectant (DMSP) in
S. meliloti.
Detection of Infectious Tobamoviruses in Forest Soils
Authors: Fillhart Ronald C. , Bachand George D. , Castello John D.
Published: December 2019

Our objectives were to evaluate elution and bait plant methods to detect infectious tobamoviruses in forest soils in New York State. Soils were collected from two forest sites: Whiteface Mountain (WF) and Heiberg Forest (HF). The effectiveness of four buffers to elute tomato mosaic tobamovirus (ToMV) from organic and mineral fractions of WF soil amended with ToMV was tested, and virus content was assessed by enzyme-linked immunosorbent assay (ELISA). The effectiveness of
Chenopodium quinoa
(Willd.) bait plants to detect the virus also was tested. Both methods then were utilized to detect tobamoviruses in 11 WF and 2 HF soil samples. A phosphate buffer (100 mM, pH 7.0) eluted more ToMV from soil than the other buffers tested. Mineral soil bound more virus than organic soil. Virus recoveries from virus-amended organic and mineral soils were 3 and 10%, respectively, and the detection sensitivity was 10 to 20 ng/g of soil. Roots of bait plants grown in all virus-amended soils tested positive by ELISA, and virus concentrations averaged 10 ng/g. Both ToMV and tobacco mosaic tobamovirus (TMV) were transmitted to
C. quinoa
by elution from one of two HF soil samples but not from the WF soil samples. A tobamovirus was detected by bait planting in 12 of 73 (16%) root extracts representing 5 of 13 soil samples (38%). Tobamovirus-like particles were seen by transmission electron microscopy in 6 of 12 infected root extracts. Tobamoviruses occur in forest soils in New York State. Abiotic soil transmission to trees may permit localized spread and persistence of these viruses in forest ecosystems.
A Small-Scale Method for Screening of Lignin-Degrading Microorganisms
Authors: Temp Ulrike , Eggert Claudia , Eriksson Karl-Erik L.
Published: December 2019

A new method to facilitate rapid screening of lignin-degrading microorganisms was developed. Fungal strains are cultivated in tissue culture plates containing
14
C-ring-labeled dehydrogenation polymerizate (DHP) (synthetic lignin). Evolved
14
CO
2
is trapped in barium-saturated filter paper and is detected by exposing the paper to X-ray film. Analysis of the autoradiograms, carried out by density measurement with an image analysis program, allows for a semiquantitative estimation of the amount of
14
CO
2
evolved. The method is especially useful for screening for new, powerful lignin-degrading strains in both man-made and natural environments. It eliminates the need for special equipment for their cultivation and trapping of
14
CO
2
as well as laborious sample analysis. The method has in this study been used to test three novel fungal isolates and a laccaseless mutant of the basidiomycete
Pycnoporus cinnabarinus
. Their ligninolytic capacities were compared with those of the potent lignin degrader
Ceriporiopsis subvermispora.
Effect of
Leuconostoc mesenteroides
NRRL B-512F Dextransucrase Carboxy-Terminal Deletions on Dextran and Oligosaccharide Synthesis
Authors: Monchois Vincent , Reverte Augustin , Remaud-Simeon Magali , Monsan Pierre , Willemot René-Marc
Published: December 2019

Dextransucrase (DSR-S) from
Leuconostoc mesenteroides
NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3′ end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the

K
m

for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.
Distribution of
Xanthomonas oryzae
pv. oryzae DNA Modification Systems in Asia
Authors: Choi Seong Ho , Vera Cruz Casiana M. , Leach Jan E.
Published: December 2019

The presence or absence of two DNA modification systems,
Xor
I and
Xor
II, in 195 strains of
Xanthomonas oryzae
pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (
Xor
I
+
Xor
II
+
,
Xor
I
+
Xor
II
−
,
Xor
I
−
Xor
II
+
and
Xor
I
−
Xor
II
−
) were detected in the population at a ratio of approximately 1:2:2:2. The
Xor
I
+
Xor
II
+
and
Xor
I
−
Xor
II
+
phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes
Xor
I
−
Xor
II
−
and
Xor
I
+
Xor
II
−
were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the
Xor
I and
Xor
II systems, we suggest that the
Xor
I modification system originated in northeast Asia and was later introduced to southeast Asia, while the
Xor
II system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of
X. oryzae
pv. oryzae that were resistant to digestion by endonuclease
Xor
II or its isoschizomer
Pvu
I also hybridized with a 7.0-kb clone that contained the
Xor
II modification system, whereas strains that were digested by
Xor
II or
Pvu
I lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in
Xor
II
+
strains within a country, indicating clonal maintenance of the
Xor
II methyltransferase gene locus. The locus was monomorphic for
X. oryzae
pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.
Molecular Analysis of a Laccase Gene from the White Rot Fungus
Pycnoporus cinnabarinus
Authors: Eggert Claudia , LaFayette Peter R. , Temp Ulrike , Eriksson Karl-Erik L. , Dean Jeffrey F. D.
Published: December 2019

It was recently shown that the white rot basidiomycete
Pycnoporus cinnabarinus
secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151–1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (
lcc3-1
) expressed by
P. cinnabarinus
during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the
P. cinnabarinus lcc3-1
product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme’s type 1 copper center. The
P. cinnabarinus lcc3-1
sequence was most similar to the sequence encoding a laccase from
Coriolus hirsutus
(level of similarity, 84%).
Truncation of Peptide Deformylase Reduces the Growth Rate and Stabilizes Solvent Production in
Clostridium beijerinckii
NCIMB 8052
Authors: Evans Victoria J. , Liyanage Hemachandra , Ravagnani Adriana , Young Michael , Kashket Eva R.
Published: December 2019

The wild-type strain of
Clostridium beijerinckii
NCIMB 8052 tends to degenerate (i.e., lose the ability to form solvents) after prolonged periods of laboratory culture. Several Tn
1545
mutants of this organism showing enhanced long-term stability of solvent production were isolated. Four of them harbor identical insertions within the
fms
(
def
) gene, which encodes peptide deformylase (PDF). The
C. beijerinckii fms
gene product contains four diagnostic residues involved in the Zn
2+
coordination and catalysis found in all PDFs, but it is unusually small, because it lacks the dispensable disordered C-terminal domain. Unlike previously characterized PDFs from
Escherichia coli
and
Thermus thermophilus
, the
C. beijerinckii
PDF can apparently tolerate N-terminal truncation. The Tn
1545
insertion in the mutants is at a site corresponding to residue 15 of the predicted gene product. This probably removes 23 N-terminal residues from PDF, leaving a 116-residue protein. The mutant PDF retains at least partial function, and it complements an
fms
(Ts) strain of
E. coli
. Northern hybridizations indicate that the mutant gene is actively transcribed in
C. beijerinckii
. This can only occur from a previously unsuspected, outwardly directed promoter located close to the right end of Tn
1545
. The Tn
1545
insertion in
fms
causes a reduction in the growth rate of
C. beijerinckii
, and, associated with this, the bacteria display an enhanced stability of solvent production. The latter phenotype can be mimicked in the wild type by reducing the growth rate. Therefore, the observed amelioration of degeneration in the mutants is probably due to their reduced growth rates.
Antibiotic Resistance Patterns of Enterococci and Occurrence of Vancomycin-Resistant Enterococci in Raw Minced Beef and Pork in Germany
Authors: Klein Günter , Pack Alexander , Reuter Gerhard
Published: December 2019

The food chain, especially raw minced meat, is thought to be responsible for an increase in the incidence of vancomycin-resistant enterococci (VRE) in human nosocomial infections. Therefore, 555 samples from 115 batches of minced beef and pork from a European Union-licensed meat-processing plant were screened for the occurrence of VRE. The processed meat came from 45 different slaughterhouses in Germany. Enterococci were isolated directly from Enterococcosel selective agar plates and also from Enterococcosel selective agar plates supplemented with 32 mg of vancomycin per liter. In addition, peptone broth was used in a preenrichment procedure, and samples were subsequently plated onto Enterococcosel agar containing vancomycin. To determine resistance, 209 isolates from 275 samples were tested with the glycopeptides vancomycin, teicoplanin, and avoparcin and 19 other antimicrobial substances by using a broth microdilution test. When the direct method was used, VRE were found in 3 of 555 samples (0.5%) at a concentration of 1.0 log CFU/g of minced meat. When the preenrichment procedure was used, 8% of the samples were VRE positive. Our findings indicate that there is a low incidence of VRE in minced meat in Germany. In addition, the resistance patterns of the VRE isolates obtained were different from the resistance patterns of clinical isolates. A connection between the occurrence of VRE in minced meat and nosocomial infections could not be demonstrated on the basis of our findings.
Oxidation of Methyl-Substituted Naphthalenes: Pathways in a Versatile
Sphingomonas paucimobilis
Strain
Authors: Dutta Tapan K. , Selifonov Sergey A. , Gunsalus Irwin C.
Published: December 2019

Aromatic compounds with alkyl substituents are abundant in fossil fuels. These compounds become important environmental sources of soluble toxic products, developmental inhibitors, etc. principally through biological activities. To assess the effect of methyl substitution on the completeness of mineralization and accumulation of pathway products, an isolate from a phenanthrene enrichment culture,
Sphingomonas paucimobilis
2322, was used. Washed cell suspensions containing cells grown on 2,6-dimethylnaphthalene in mineral medium were incubated with various mono-, di-, and trimethylnaphthalene isomers, and the products were identified and quantified by gas chromatography and mass spectrometry. The data revealed enzymes with relaxed substrate specificity that initiate metabolism either by methyl group monoxygenation or by ring dioxygenation. Congeners with a methyl group on each ring initially hydroxylate a methyl, and this is followed by conversion to a carboxyl; when there are two methyl groups on a single ring, the first reaction is aryl dioxygenation of the unsubstituted ring. Intermediates are channeled to primary ring fission via dihydrodiols to form methyl-substituted salicylates. Further evidence that there are multiple pathways comes from the fact that both phthalate and (methyl)salicylate are formed from 2-methylnaphthalene.
Rapid Benzene Degradation in Methanogenic Sediments from a Petroleum-Contaminated Aquifer
Authors: Weiner Jonathan M. , Lovley Derek R.
Published: December 2019

In methanogenic sediments from a petroleum-contaminated aquifer, [
14
C]benzene was converted to
14
CH
4
and
14
CO
2
without an apparent lag. Phenol, acetate, and propionate were intermediates in benzene mineralization. These results suggest that alternative electron acceptors need not be available for there to be significant natural attenuation of benzene in some petroleum-contaminated aquifers.
Structures of Homologous Composite Transposons Carrying
cbaABC
Genes from Europe and North America
Authors: Di Gioia Diana , Peel Michelle , Fava Fabio , Wyndham R. Campbell
Published: December 2019

IS
1071
is a class II transposable element carrying a
tnpA
gene related to the transposase genes of the Tn
3
family. Copies of IS
1071
that are conserved with more than 99% nucleotide sequence identity have been found as direct repeats flanking a remarkable variety of catabolic gene sequences worldwide. The sequences of chlorobenzoate catabolic transposons found on pBRC60 (Tn
5271
) in Niagara Falls, N.Y., and on pCPE3 in Bologna, Italy, show that these transposons were formed from highly homologous IS
1071
and
cbaABC
components (levels of identity, >99.5 and >99.3%, respectively). Nevertheless, the junction sequences between the IS
1071L
and IS
1071R
elements and the internal DNA differ by 41 and 927 bp, respectively, suggesting that these transposons were assembled independently on the two plasmids. The formation of the right junction in both transposons truncated an open reading frame for a putative aryl-coenzyme A ligase with sequence similarity to benzoate- and
p
-hydroxybenzoate-coenzyme A ligases of
Rhodopseudomonas palustris.
Diversity of European Lyme Disease Spirochetes at the Southern Margin of Their Range
Authors: Matuschka Franz-Rainer , Klug Birte , Schinkel Thomas W. , Spielman Andrew , Richter Dania
Published: December 2019

We determined whether the genospecies diversity of Lyme disease spirochetes in vector ticks questing on a subtropical island is as broad as that in Central Europe. Although spirochetes infected <1% of the ticks sampled on Madeira Island, these infections included all three genospecies implicated in human disease. Therefore, spirochetal diversity is as great at the southern margin as it is in the center of this pathogen’s range.
Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader,
Rhodococcus
sp. Strain RHA1
Authors: Yamada Akihiro , Kishi Hidekazu , Sugiyama Katsumi , Hatta Takashi , Nakamura Kanji , Masai Eiji , Fukuda Masao
Published: December 2019

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes,
etbD1
and
etbD2
, were cloned from a strong polychlorinated biphenyl (PCB) degrader,
Rhodococcus
sp. strain RHA1, and their nucleotide sequences were determined. The
etbD2
gene was located in the vicinity of
bphA
gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the
etbD2
gene fragment as a probe, we cloned the
etbD1
gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in α/β hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as
dmpD
,
cumD
,
todF
, and
xylF
, and not very similar to the amino acid sequences of the products of
bphD
genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene,
bphD
, were expressed in
Escherichia coli
, and their relative enzymatic activities were examined. The product of
bphD
was very specific to HPDA, and the products of
etbD1
and
etbD2
were specific to HOHD. All of the gene products exhibited poor activities against the
meta
-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and
ortho
-xylene. Strain RCD1, an RHA1 mutant strain lacking both the
bphD
gene and the
etbD2
gene, grew well on ethylbenzene. This result suggested that the
etbD1
gene product is involved in the
meta
-cleavage metabolic pathway of ethylbenzene.
The
gal
Genes for the Leloir Pathway of
Lactobacillus casei
64H
Authors: Bettenbrock Katja , Alpert Carl-Alfred
Published: December 2019

The
gal
genes from the chromosome of
Lactobacillus casei
64H were cloned by complementation of the
galK2
mutation of
Escherichia coli
HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA
Hin
dIII fragment. Detailed studies with other
E. coli
K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of the
L. casei gal
operon is
galKETRM
. Because parts of the gene for the mutarotase as well as the promoter region upstream of
galK
were not cloned on pKBL1, the regions flanking the
Hin
dIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the
gal
genes constitute an operon that is transcribed from two promoters. The
galKp
promoter is inducible by galactose in the medium, while
galEp
constitutes a semiconstitutive promoter located in
galK
.
Female Fertility and Mating Type Distribution in a South African Population of
Fusarium subglutinans
f. sp.
pini
Authors: Britz H. , Wingfield M. J. , Coutinho T. A. , Marasas W. F. O. , Leslie J. F.
Published: December 2019

Fusarium subglutinans
f. sp.
pini
is the causal agent of pitch canker disease of pines. The initial occurrence of
F. subglutinans
f. sp.
pini
in South Africa was limited to a single nursery, and isolates from this population are capable of reproducing sexually. We determined the effective population number,

N
e

, of this population by using mating type and male/hermaphrodite polymorphisms as indicators. The effective population number for mating type,

N
e(mt)

, is 99% of the count (total population), and that for male/hermaphrodite status,

N
e(f)

, is 42 to 46% of the count (total population). The number of strains that can function as the female parent limits the effective population number of this population. If this population is stable, then, depending upon assumptions about mutation and selection, sexual reproduction need occur only once per 26 to 153 asexual generations to maintain this level of sexual fertility.
Characterization of Metabolites in the Biotransformation of 2,4,6-Trinitrotoluene with Anaerobic Sludge: Role of Triaminotoluene
Authors: Hawari Jalal , Halasz A. , Paquet L. , Zhou E. , Spencer B. , Ampleman G. , Thiboutot S.
Published: December 2019

The present study describes the biotransformation of 2,4,6-trinitrotoluene (TNT) (220 μM) by using anaerobic sludge (10%, vol/vol) supplemented with molasses (3.3 g/liter). Despite the disappearance of TNT in less than 15 h, roughly 0.1% of TNT was attributed to mineralization (
14
CO
2
). A combination of solid-phase microextraction–gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry identified two distinctive cycles in the degradation of TNT. One cycle was responsible for the stepwise reduction of TNT to eventually produce triaminotoluene (TAT) in relatively high yield (160 μM). The other cycle involved TAT and was responsible for the production of azo derivatives, e.g., 2,2′,4,4′-tetraamino-6,6′-azotoluene (2,2′,4,4′-TA-6,6′-azoT) and 2,2′,6,6′-tetraamino-4,4′-azotoluene (2,2′,6,6′-TA-4,4′-azoT) at pH 7.2. These azo compounds were also detected when TAT was treated with the anaerobic sludge but not with an autoclaved sludge, suggesting the biotic nature of their formation. When the anaerobic conditions in the TAT-containing culture medium were removed by aeration and/or acidification (pH 3), the corresponding phenolic compounds, e.g., hydroxy-diaminotoluenes and dihydroxy-aminotoluenes, were observed at room temperature. Trihydroxytoluene was detected only after heating TAT in water at 100°C. When
13
CH
3
-labeled TNT was used as the N source in the above microcosms, we were unable to detect
13
C-labeled
p
-cresol or [
13
CH
3
]toluene, indicating the absence of denitration or deamination in the biodegradation process. The formation and disappearance of TAT were not accompanied by mineralization, suggesting that TAT acted as a dead-end metabolite.
Randomly Amplified Polymorphic DNA PCR Analysis of Bovine
Cryptosporidium parvum
Strains Isolated from the Watershed of the Red River of the North
Authors: Shianna Kevin V. , Rytter Russell , Spanier Jonathan G.
Published: December 2019

Cryptosporidium parvum
is a protozoan parasite that causes the disease cryptosporidiosis in a variety of mammals, including neonatal calves and humans. Millions of oocysts are shed during acute cryptosporidiosis, and zoonotic transmission is inferred, though not proven, to be a general phenomenon. Very little is known about the degree of strain variation exhibited by bovine and human isolates, though such knowledge would enable the amount of bovine-to-human transmission to be more precisely analyzed. This research was initiated to determine whether variations exist among bovine strains isolated from a localized geographic area, the watershed of the Red River of the North. Sixteen strains were isolated and compared to each other and to two human and two calf strains from Australia by randomly amplified polymorphic DNA PCR. A statistical analysis of the data indicated that the isolates belonged to four different groups of strains.