bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.