bluuebunny/crossref_metadata_2025 huggingface dataset:
Longitudinal Study ofEscherichia coliO157:H7 Dissemination on Four Dairy Farms in Wisconsin
Authors: Shere J. A. , Bartlett K. J. , Kaspar C. W.
Published: December 2019

A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination ofEscherichia coliO157:H7. A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months). Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative. Farm R had two separate periods ofE. coliO157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996). Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 × 102to 8.7 × 104CFU per g.E. coliO157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X. When present in animal drinking water,E. coliO157:H7 disseminated through the cohort cattle and other cattle that used the water source.E. coliO157:H7 was found in water at <1 to 23 CFU/ml. Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm. The isolates from farm R displayed two predominantXbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively. Six additional REDP that were ≥89% similar to REDP 3 or REDP 7 were identified among the farm R isolates. Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3. Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9. These results suggest thatE. coliO157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period.
Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum
Authors: Schwan Rosane Freitas
Published: December 2019

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast,
Saccharomyces cerevisiae
var.
chevalieri
, two lactic acid bacterial species,
Lactobacillus lactis
and
Lactobacillus plantarum
, and two acetic acid bacterial species,
Acetobacter aceti
and
Gluconobacter oxydans
subsp.
suboxydans
. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.
Targeted Mutants of
Cochliobolus carbonum
Lacking the Two Major Extracellular Polygalacturonases
Authors: Scott-Craig John S. , Cheng Yi-Qiang , Cervone Felice , De Lorenzo Giulia , Pitkin John W. , Walton Jonathan D.
Published: December 2019

The filamentous fungus
Cochliobolus carbonum
produces endo-α1,4-polygalacturonase (endoPG), exo-α1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a
pgn1
mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG,
PGX1
, was isolated with PCR primers based on peptide sequences from the protein. The product of
PGX1
, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus
Aspergillus tubingensis
. Strains of
C. carbonum
mutated in
PGX1
were constructed by targeted gene disruption and by gene replacement. Growth of
pgx1
mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double
pgn1/pgx1
mutant strain was constructed by crossing. The double mutant grew as well as the
pgx1
single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the
pgn1/pgx1
mutant on pectin could be due to one or more of these residual activities.
Recovery of Humic-Reducing Bacteria from a Diversity of Environments
Authors: Coates John D. , Ellis Debra J. , Blunt-Harris Elizabeth L. , Gaw Catherine V. , Roden Eric E. , Lovley Derek R.
Published: December 2019

To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family
Geobacteraceae
. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the
Geobacteraceae
. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that
Geobacteraceae
species might be important humic-reducing organisms in sediments.
Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation
Authors: Huang Ching-Tsan , Xu Karen D. , McFeters Gordon A. , Stewart Philip S.
Published: December 2019

The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both
Klebsiella pneumoniae
and
Pseudomonas aeruginosa
biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in
K. pneumoniae
colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In
K. pneumoniae
biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in
P. aeruginosa
biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.
High-Pressure Inactivation and Sublethal Injury of Pressure-Resistant
Escherichia coli
Mutants in Fruit Juices
Authors: Garcia-Graells Cristina , Hauben Kristel J. A. , Michiels Chris W.
Published: December 2019

The potential of high-pressure-resistant mutants of
Escherichia coli
to survive high-pressure pasteurization in fruit juices and in low-pH buffers was investigated. Treatments with up to 500 MPa of pressure caused only a limited direct inactivation of the mutants but resulted in an accelerated low-pH inactivation during subsequent storage.
Sonication-Dependent Electroporation of the Erythromycin-Producing Bacterium
Saccharopolyspora erythraea
Authors: Fitzgerald Nancy B. , English R. Samuel , Lampel Jay S. , Vanden Boom Thomas J.
Published: December 2019

We report the development of an electrotransformation method applicable to all strains of
Saccharopolyspora erythraea
examined to date. Vegetatively grown mycelia were rendered electrocompetent by subjecting mycelial suspensions to ultrasound pulses. The protocol provides an alternative route for the introduction of DNA into filamentous microorganisms otherwise recalcitrant to transformation techniques.
Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer
Authors: Vester Flemming , Ingvorsen Kjeld
Published: December 2019

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (
35
SO
4
2−
). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a
35
S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO
4
2−
) of 10
−14
to 10
−13
mol of SO
4
2−
cell
−1
day
−1
were calculated, which is within the range of qSO
4
2−
values previously reported for pure cultures of SRB (10
−15
to 10
−14
mol of SO
4
2−
cell
−1
day
−1
). qSO
4
2−
values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10
−10
to 7 × 10
−10
mol of SO
4
2−
cell
−1
day
−1
), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.
Starvation-Induced Changes in Motility, Chemotaxis, and Flagellation of
Rhizobium meliloti
Authors: Wei Xueming , Bauer Wolfgang D.
Published: December 2019

The changes in motility, chemotactic responsiveness, and flagellation of
Rhizobium meliloti
RMB7201, L5-30, and JJ1c10 were analyzed after transfer of the bacteria to buffer with no available C, N, or phosphate. Cells of these three strains remained viable for weeks after transfer to starvation buffer (SB) but lost all motility within just 8 to 72 h after transfer to SB. The rates of motility loss differed by severalfold among the strains. Each strain showed a transient, two- to sixfold increase in chemotactic responsiveness toward glutamine within a few hours after transfer to SB, even though motility dropped substantially during the same period. Strains L5-30 and JJ1c10 also showed increased responsiveness to the nonmetabolizable chemoattractant cycloleucine. Cycloleucine partially restored the motility of starving cells when added after transfer and prevented the loss of motility when included in the SB used for initial suspension of the cells. Thus, interactions between chemoattractants and their receptors appear to affect the regulation of motility in response to starvation independently of nutrient or energy source availability. Electron microscopic observations revealed that
R. meliloti
cells lost flagella and flagellar integrity during starvation, but not as fast, nor to such a great extent, as the cells lost motility. Even after prolonged starvation, when none of the cells were actively motile, about one-third to one-half of the initially flagellated cells retained some flagella. Inactivation of flagellar motors therefore appears to be a rapid and important response of
R. meliloti
to starvation conditions. Flagellar-motor inactivation was at least partially reversible by addition of either cycloleucine or glucose. During starvation, some cells appeared to retain normal flagellation, normal motor activity, or both for relatively long periods while other cells rapidly lost flagella, motor activity, or both, indicating that starvation-induced regulation of motility may proceed differently in various cell subpopulations.
A Mixed Culture Recovery Method Indicates that Enteric Bacteria Do Not Enter the Viable but Nonculturable State
Authors: Bogosian Gregg , Morris Patricia J. L. , O’Neil Julia P.
Published: December 2019

A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains of
Escherichia coli
,
Klebsiella pneumoniae
,
Enterococcus faecalis
,
Enterobacter aerogenes
, and
Salmonella choleraesuis
, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.
Germination, Growth, and Sporulation of
Bacillus thuringiensis
subsp.
israelensis
in Excreted Food Vacuoles of the Protozoan
Tetrahymena pyriformis
Authors: Manasherob Robert , Ben-Dov Eitan , Zaritsky Arieh , Barak Ze’ev
Published: December 2019

Spores of
Bacillus thuringiensis
subsp.
israelensis
and their toxic crystals are bioencapsulated in the protozoan
Tetrahymena pyriformis
, in which the toxin remains stable. Each
T. pyriformis
cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of
B. thuringiensis
subsp.
israelensis
was observed after 7 h as filaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of
B. thuringiensis
subsp.
israelensis
-killed larvae and pupae, but this bioencapsulation represents a new possible mode of
B. thuringiensis
subsp.
israelensis
recycling in nontarget organisms.
Brominated Biphenyls Prime Extensive Microbial Reductive Dehalogenation of Aroclor 1260 in Housatonic River Sediment
Authors: Bedard Donna L. , Van Dort Heidi , Deweerd Kim A.
Published: December 2019

The upper Housatonic River and Woods Pond (Lenox, Mass.), a shallow impoundment on the river, are contaminated with polychlorinated biphenyls (PCBs), the residue of partially dechlorinated Aroclor 1260. Certain PCB congeners have the ability to activate or “prime” anaerobic microorganisms in Woods Pond sediment to reductively dehalogenate the Aroclor 1260 residue. We proposed that brominated biphenyls might have the same effect and tested the priming activities of 14 mono-, di-, and tribrominated biphenyls (350 μM) in anaerobic microcosms of sediment from Woods Pond. All of the brominated biphenyls were completely dehalogenated to biphenyl, and 13 of them primed PCB dechlorination. Measured in terms of chlorine removal and decrease in the proportion of hexa- through nonachlorobiphenyls, the microbial PCB dechlorination primed by several brominated biphenyls was nearly twice as effective as that primed by chlorinated biphenyls. Congeners containing a
meta
bromine primed Dechlorination Process N (flanked
meta
dechlorination), and congeners containing an unflanked
para
bromine primed Dechlorination Process P (flanked
para
dechlorination). Two
ortho
-substituted congeners, 2-bromobiphenyl and 2,6-dibromobiphenyl (2-BB and 26-BB), also primed Process N dechlorination. The most effective primers were 26-BB, 245-BB, 25-3-BB, and 25-4-BB. The microbial dechlorination primed by 26-BB converted ∼75% of the hexa- through nonachlorobiphenyls to tri- and tetrachlorobiphenyls in 100 days and removed ∼75% of the PCBs that are most persistent in humans. These results represent a major step toward identifying an effective method for accelerating PCB dechlorination in situ. The challenge now is to identify naturally occurring compounds that are safe and effective primers.
Molecular Analysis of the Locus Responsible for Production of Plantaricin S, a Two-Peptide Bacteriocin Produced by
Lactobacillus plantarum
LPCO10
Authors: Stephens Sarah K. , Floriano Belén , Cathcart Declan P. , Bayley Susan A. , Witt Valerie F. , Jiménez-Díaz Rufino , Warner Philip J. , Ruiz-Barba José Luis
Published: December 2019

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by
Lactobacillus plantarum
LPCO10 (R. Jiménez-Dı́az, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459–4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated
plsA
and
plsB
and encoding peptides α and β, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of
plsA
and
plsB
revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene
agrB
from
Staphylococcus aureus
, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an
agr
-like regulatory system for the production of plantaricin S is postulated.
Chloromethane Metabolism by
Methylobacterium
sp. Strain CM4
Authors: Vannelli Todd , Studer Alex , Kertesz Michael , Leisinger Thomas
Published: December 2019

Methylobacterium
sp. strain CM4 metabolized chloromethane quantitatively with a molar yield of 2.8 g of whole-cell protein/mol of C. This value was similar to that observed after growth with methanol (2.9 g of protein/mol of C) and about three times larger than the yield with formate (0.94 g of protein/mol of C). Chloromethane dehalogenation activity was inducible. MiniTn
5
transposon insertion mutants with altered growth characteristics with chloromethane and other C
1
compounds were isolated and characterized. Nine of these were unable to grow with chloromethane but were able to grow with methanol, methylamine, or formate. Seventy-three transposon mutants that were defective in the utilization of either methanol, methylamine, methanol plus methylamine, or formate could still grow with chloromethane. Based on the protein yield data and the properties of the transposon mutants, we propose a pathway for chloromethane metabolism that depends on methyltransferase and dehydrogenase activities.
Effect of Dietary Stress on Fecal Shedding of
Escherichia coli
O157:H7 in Calves
Authors: Cray William C. , Casey Thomas A. , Bosworth Brad T. , Rasmussen Mark A.
Published: December 2019

Two groups of calves were subjected to dietary stress by withholding of food beginning 1 or 14 days after inoculation with 10
10
CFU of
Escherichia coli
O157:H7. Following treatment, neither group had a significant increase in fecal shedding of
E. coli
O157:H7. A third group of calves had food withheld for 48 h prior to inoculation with 10
7
CFU of
E. coli
O157:H7. These calves were more susceptible to infection and shed significantly more
E. coli
O157:H7 organisms than calves maintained on a normal diet.
Reduction of the 2,2′-Azinobis(3-Ethylbenzthiazoline-6-Sulfonate) Cation Radical by Physiological Organic Acids in the Absence and Presence of Manganese
Authors: Collins Patrick J. , Dobson Alan D. W. , Field Jim A.
Published: December 2019

Laccase is a copper-containing phenoloxidase, involved in lignin degradation by white rot fungi. The laccase substrate range can be extended to include nonphenolic lignin subunits in the presence of a noncatalytic cooxidant such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), with ABTS being oxidized to the stable cation radical, ABTS
·+
, which accumulates. In this report, we demonstrate that the ABTS
·+
can be efficiently reduced back to ABTS by physiologically occurring organic acids such as oxalate, glyoxylate, and malonate. The reduction of the radical by oxalate results in the formation of H
2
O
2
, indicating the formation of O
2
·−
as an intermediate. O
2
·−
itself was shown to act as an ABTS
·+
reductant. ABTS
·+
reduction and H
2
O
2
formation are strongly stimulated by the presence of Mn
2+
, with accumulation of Mn
3+
being observed. Additionally, 4-methyl-
O
-isoeugenol, an unsaturated lignin monomer model, is capable of directly reducing ABTS
·+
. These data suggest several mechanisms for the reduction of ABTS
·+
which would permit the effective use of ABTS as a laccase cooxidant at catalytic concentrations.
Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniques
Authors: Fuller Nicholas J. , Wilson William H. , Joint Ian R. , Mann Nicholas H.
Published: December 2019

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus
Synechococcus
. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.
Development of a Genetic Transformation System for an Alga-Lysing Bacterium
Authors: Kato Junichi , Amie Junya , Murata Yoshinori , Kuroda Akio , Mitsutani Atsushi , Ohtake Hisao
Published: December 2019

Four marine bacteria,
Alteromonas
sp. strains A27, A28, A29, and A30, that lyse the diatom
Skeletonema costatum
NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms
Thalassiosira
sp. and
Eucampia zodiacs
and the raphidophycean flagellate
Chattonella antiqua
. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in
Alteromonas
sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in
Escherichia coli
and
Alteromonas
sp. strain A28 was constructed by fusing pAS28 and
E. coli
vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10
6
transformants per μg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb
Eco
RI-
Hin
dIII region of pAS28 was essential for plasmid maintenance in strain A28. This
Eco
RI-
Hin
dIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.
Genotypic Characterization of
Bradyrhizobium
Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing
Authors: Vinuesa Pablo , Rademaker Jan L. W. , de Bruijn Frans J. , Werner Dietrich
Published: December 2019

We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (
Chamaecytisus proliferus
) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains,
Bradyrhizobium japonicum
USDA 110
spc
4 and
Bradyrhizobium
sp. strain (
Centrosema
) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the
B. japonicum/Rhodopseudomonas
rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated
Glycine max
or
Leucaena leucocephala
, but all nodulated
Acacia pendula
,
C. proliferus
,
Macroptilium atropurpureum
, and
Vigna unguiculata
.
Role of
Streptococcus thermophilus
MR-1C Capsular Exopolysaccharide in Cheese Moisture Retention
Authors: Low Deborah , Ahlgren Jeffrey A. , Horne Diane , McMahon Donald J. , Oberg Craig J. , Broadbent Jeffery R.
Published: December 2019

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair,
Streptococcus thermophilus
MR-1C and
Lactobacillus delbrueckii
subsp.
bulgaricus
MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799–805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains
S. thermophilus
TA061 and
Lactobacillus helveticus
LH100 showed that
S. thermophilus
MR-1C was responsible for the increased cheese moisture level. To investigate the role of the
S. thermophilus
MR-1C EPS in cheese moisture retention, the
epsE
gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with
L. helveticus
LH100 plus the non-EPS-producing mutant
S. thermophilus
DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the
S. thermophilus
MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of
d
-galactose,
l
-rhamnose, and
l
-fucose in a ratio of 5:2:1.
Culture Conditions Control Expression of the Genes for Aflatoxin and Sterigmatocystin Biosynthesis in
Aspergillus parasiticus
and
A. nidulans
Authors: Feng Guo Hong , Leonard Thomas J.
Published: December 2019

High temperature and nitrate supported gene expression for sterigmatocystin biosynthesis in
Aspergillus nidulans
; ammonium did not. Homologous genes for aflatoxin biosynthesis in
A. parasiticus
showed the opposite transcript expression pattern, suggesting that the two mycotoxins are regulated differently. The
aflR
gene is postulated to require additional genetic elements to effect its own activation by the different culture conditions. A patulin polyketide synthase (PKS) gene was found to be regulated differently than the aflatoxin PKS. Thus, the biosyntheses of structurally similar compounds in these two fungi appear to be regulated very differently.
Detection of
Toxoplasma gondii
Oocysts in Drinking Water
Authors: Isaac-Renton Judith , Bowie William R. , King Arlene , Irwin G. Stewart , Ong Corinne S. , Fung C. P. , Shokeir M. Omar , Dubey J. P.
Published: December 2019

The world’s largest outbreak of waterborne toxoplasmosis occurred in a municipality in the western Canadian province of British Columbia. When drinking water emerged as a possible source of infection during the outbreak investigation, a laboratory method was needed to attempt detection of the parasite,
Toxoplasma gondii
. The method developed was based on the current U.S. Environmental Protection Agency method for detection of
Cryptosporidium
oocysts. Collection of large-volume drinking water samples and cartridge filter processing were unchanged, although identification of
Toxoplasma
oocysts in the filter retentate was carried out by using a previously described rodent model. Validation of the method developed was tested by using oocysts from a well-characterized
Toxoplasma
strain.
Evaluation of Lacticin 3147 and a Teat Seal Containing This Bacteriocin for Inhibition of Mastitis Pathogens
Authors: Ryan Máire P. , Meaney William J. , Ross R. Paul , Hill Colin
Published: December 2019

Lacticin 3147 is a broad-spectrum bacteriocin produced byLactococcus lactissubsp.lactisDPC3147 which is bactericidal against a range of mastitis-causing streptococci and staphylococci. In this study, both lacticin 3147 and the lantibiotic nisin were separately incorporated into an intramammary teat seal product. The seal containing lacticin 3147 exhibited excellent antimicrobial activity and might form the basis of an improved treatment for the prevention of mastitis in dry cows.
Epidemiological Typing of
Campylobacter
Isolates from Meat Processing Plants by Pulsed-Field Gel Electrophoresis, Fatty Acid Profile Typing, Serotyping, and Biotyping
Authors: Steele M. , McNab B. , Fruhner L. , DeGrandis S. , Woodward D. , Odumeru J. A.
Published: December 2019

Campylobacter
spp. are a leading cause of bacterial gastroenteritis. Foods of animal origin, particularly undercooked poultry, are common sources of
Campylobacter
species associated with disease in humans. A collection of 110
Campylobacter jejuni
and 31
C. coli
human and environmental isolates from different Ontario, Canada, abattoirs were analyzed by pulsed-field gel electrophoresis, fatty acid profile typing, and biotyping. Previously collected serotyping data for the same isolates were also analyzed in this study. Pulsed-field gel electrophoresis was found to be the most discriminatory of the typing methods, followed by serotyping, fatty acid profile typing, and biotyping. A wide variety of typing profiles were observed within the isolates, suggesting that several different
Campylobacter
sp. strains were present within the abattoirs.
Kinetic Parameters of Denitrification in a River Continuum
Authors: García-Ruiz Roberto , Pattinson Sarah N. , Whitton Brian A.
Published: December 2019

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (Kmap) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (Vmaxap) ranged between 35.8 and 324 μmol of N m−2h−1in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m−2h−1). A study of nitrous oxide (N2O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N2O-N m−2h−1) at the headwater site to 27 μmol of N2O-N m−2h−1at the downstream site. In the Wiske the rate was up to 570 μmol of N2O-N m−2h−1, accounting for up to 80% of total N gas production.
Seasonal and Spatial Variability of Bacterial and Archaeal Assemblages in the Coastal Waters near Anvers Island, Antarctica
Authors: Murray A. E. , Preston C. M. , Massana R. , Taylor L. T. , Blakis A. , Wu K. , DeLong E. F.
Published: December 2019

A previous report of high levels of members of the domain
Archaea
in Antarctic coastal waters prompted us to investigate the ecology of Antarctic planktonic prokaryotes. rRNA hybridization techniques and denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial V3 region were used to study variation in Antarctic picoplankton assemblages. In Anvers Island nearshore waters during late winter to early spring, the amounts of archaeal rRNA ranged from 17.1 to 3.6% of the total picoplankton rRNA in 1996 and from 16.0 to 1.0% of the total rRNA in 1995. Offshore in the Palmer Basin, the levels of archaeal rRNA throughout the water column were higher (average, 24% of the total rRNA) during the same period in 1996. The archaeal rRNA levels in nearshore waters followed a highly seasonal pattern and markedly decreased during the austral summer at two stations. There was a significant negative correlation between archaeal rRNA levels and phytoplankton levels (as inferred from chlorophyll
a
concentrations) in nearshore surface waters during the early spring of 1995 and during an 8-month period in 1996 and 1997. In situ hybridization experiments revealed that 5 to 14% of DAPI (4′,6-diamidino-2-phenylindole)-stained cells were archaeal, corresponding to 0.9 × 10
4
to 2.7 × 10
4
archaeal cells per ml, in late winter 1996 samples. Analysis of bacterial ribosomal DNA fragments by DGGE revealed that the assemblage composition may reflect changes in water column stability, depth, or season. The data indicate that changes in Antarctic seasons are accompanied by significant shifts in the species composition of bacterioplankton assemblages and by large decreases in the relative proportion of archaeal rRNA in the nearshore water column.
Trifolitoxin Production Increases Nodulation Competitiveness of
Rhizobium etli
CE3 under Agricultural Conditions
Authors: Robleto Eduardo A. , Kmiecik Kenneth , Oplinger Edward S. , Nienhuis James , Triplett Eric W.
Published: December 2019

A major barrier to the use of nitrogen-fixing inoculum strains for the enhancement of legume productivity is the inability of commercially available strains to compete with indigenous rhizobia for nodule formation. Despite extensive research on nodulation competitiveness, there are no examples of field efficacy studies of strains that have been genetically improved for nodulation competitiveness. We have shown previously that production of the peptide antibiotic trifolitoxin (TFX) by
Rhizobium etli
results in significantly increased nodule occupancy values in nonsterile soil in growth chamber experiments (E. A. Robleto, A. J. Scupham, and E. W. Triplett, Mol. Plant-Microbe Interact. 10:228–233, 1997). To determine whether TFX production by
Rhizobium etli
increases nodulation competitiveness in field-grown plants, seeds of
Phaseolus vulgaris
were inoculated with mixtures of
Rhizobium etli
strains at different ratios. The three nearly isogenic inoculum strains used included TFX-producing and non-TFX-producing strains, as well as a TFX-sensitive reference strain. Data was obtained over 2 years for nodule occupancy and over 3 years for assessment of the effect of the TFX production phenotype on grain yield. In comparable mixtures in which the test strain accounted for between 5 and 50% of the inoculum, the TFX-producing strain exhibited at least 20% greater nodule occupancy than the non-TFX-producing strain in both years. The TFX production phenotype had no effect on grain yield over 3 years; the average yields reached 2,400 kg/ha. These results show that addition of the TFX production phenotype significantly increases nodule occupancy under field conditions without adverse effects on grain yield. As we used common inoculation methods in this work, there are no practical barriers to the commercial adoption of the TFX system for agriculture.
Increased Carotenoid Production by the Food Yeast
Candida utilis
through Metabolic Engineering of the Isoprenoid Pathway
Authors: Shimada Hiroshi , Kondo Keiji , Fraser Paul D. , Miura Yutaka , Saito Toshiko , Misawa Norihiko
Published: December 2019

The yeast
Candida utilis
does not possess an endogenous biochemical pathway for the synthesis of carotenoids. The central isoprenoid pathway concerned with the synthesis of prenyl lipids is present in
C. utilis
and active in the biosynthesis of ergosterol. In our previous study, we showed that the introduction of exogenous carotenoid genes,
crtE
,
crtB
, and
crtI
, responsible for the formation of lycopene from the precursor farnesyl pyrophosphate, results in the
C. utilis
strain that yields lycopene at 1.1 mg per g (dry weight) of cells (Y. Miura, K. Kondo, T. Saito, H. Shimada, P. D. Fraser, and N. Misawa, Appl. Environ. Microbiol. 64:1226–1229, 1998). Through metabolic engineering of the isoprenoid pathway, a sevenfold increase in the yield of lycopene has been achieved. The influential steps in the pathway that were manipulated were 3-hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase, encoded by the
HMG
gene, and squalene synthase, encoded by the
ERG9
gene. Strains overexpressing the
C. utilis
HMG-CoA reductase yielded lycopene at 2.1 mg/g (dry weight) of cells. Expression of the HMG-CoA catalytic domain alone gave 4.3 mg/g (dry weight) of cells; disruption of the
ERG9
gene had no significant effect, but a combination of
ERG9
gene disruption and the overexpression of the HMG catalytic domain yielded lycopene at 7.8 mg/g (dry weight) of cells. The findings of this study illustrate how modifications in related biochemical pathways can be utilized to enhance the production of commercially desirable compounds such as carotenoids.
Specific Detection of
Legionella pneumophila
: Construction of a New 16S rRNA-Targeted Oligonucleotide Probe
Authors: Grimm Dorothee , Merkert Hilde , Ludwig Wolfgang , Schleifer Karl-Heinz , Hacker Jörg , Brand Bettina C.
Published: December 2019

Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of
Legionella pneumophila
which allows the differentiation of
L. pneumophila
from other
Legionella
species without cultivation. The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of
L. pneumophila
, five different
Legionella
spp. and three nonlegionella species as reference strains. Furthermore,
L. pneumophila
cells could be easily distinguished from
Legionella micdadei
and
Pseudomonas aeruginosa
cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan
Acanthamoeba castellanii
.
Giardia
sp. Cysts and Infectious
Cryptosporidium parvum
Oocysts in the Feces of Migratory Canada Geese (
Branta canadensis
)
Authors: Graczyk Thaddeus K. , Fayer Ronald , Trout James M. , Lewis Earl J. , Farley C. Austin , Sulaiman Irshad , Lal Altaf A.
Published: December 2019

Fecal droppings of migratory Canada geese,
Branta canadensis
, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of
Cryptosporidium parvum
and
Giardia
spp.
Cryptosporidium
sp. oocysts were found in feces at seven of nine sites, and
Giardia
cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of
Cryptosporidium parvum
. Waterfowl can disseminate infectious
C. parvum
oocysts in the environment.
Use of the Lactococcal
nisA
Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength
Authors: Eichenbaum Zehava , Federle Michael J. , Marra Diana , de Vos Willem M. , Kuipers Oscar P. , Kleerebezem Michiel , Scott June R.
Published: December 2019

We characterized the regulated activity of the lactococcal
nisA
promoter in strains of the gram-positive species
Streptococcus pyogenes
,
Streptococcus agalactiae
,
Streptococcus pneumoniae
,
Enterococcus faecalis
, and
Bacillus subtilis. nisA
promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the
nisA
promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the
nisA
promoter was compared to the efficiencies of the
Spac
,
xylA
, and
lacA
promoters in
B. subtilis
and in
S. pyogenes
. Because
nisA
promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.
Transformation of Industrial Dyes by Manganese Peroxidases from
Bjerkandera adusta
and
Pleurotus eryngii
in a Manganese-Independent Reaction
Authors: Heinfling A. , Martínez M. J. , Martínez A. T. , Bergbauer M. , Szewzyk U.
Published: December 2019

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from
Bjerkandera adusta
and other white rot fungi. The dyes were not oxidized or were oxidized very little by
Phanerochaete chrysosporium
manganese peroxidase (MnP) or by a chemically generated Mn
3+
-lactate complex. Lignin peroxidase (LiP) from
B. adusta
also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The
B. adusta
and
Pleurotus eryngii
MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn
2+
. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn
2+
-independent manner. The reactions with the dyes were characterized by apparent

K
m

values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn
2+
, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn
3+
-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by
B. adusta
MnP1.