bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Basic Replicon of
Rhodococcus
Plasmid pSOX and Development of a
Rhodococcus-Escherichia coli
Shuttle Vector
Authors: Denis-Larose Claude , Bergeron Hélène , Labbé Diane , Greer Charles W. , Hawari Jalal , Grossman Matthew J. , Sankey Bruce M. , Lau Peter C. K.
Published: December 2019

The replication region of a 100-kb desulfurization plasmid (pSOX) from
Rhodococcus
sp. strain X309 was localized to a 4-kb
Kpn
I fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the
Escherichia coli
T7 polymerase/promoter system. In
E. coli
, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS−, and the chloramphenicol resistance (Cm
r
) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the
Bacillus subtilis sacB
gene in a strain of
Rhodococcus
, rendering it sensitive to the presence of sucrose.
Identification of a Marine AgarolyticPseudoalteromonasIsolate and Characterization of Its Extracellular Agarase
Authors: Vera Jorge , Alvarez Raul , Murano Erminio , Slebe Juan Carlos , Leon Oscar
Published: December 2019

The phenotypic and agarolytic features of an unidentified marine bacteria that was isolated from the southern Pacific coast was investigated. The strain was gram negative, obligately aerobic, and polarly flagellated. On the basis of several phenotypic characters and a phylogenetic analysis of the genes coding for the 16S rRNA, this strain was identified asPseudoalteromonas antarcticastrain N-1. In solid agar, this isolate produced a diffusible agarase that caused agar softening around the colonies. An extracellular agarase was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on DEAE-cellulose. The purified protein was determined to be homogeneous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it had a molecular mass of 33 kDa. The enzyme hydrolyzed the β-1,4-glycosydic linkages of agar, yielding neoagarotetraose and neoagarohexaose as the main products, and exhibited maximal activity at pH 7. The enzyme was stable at temperatures up to 30°C, and its activity was not affected by salt concentrations up to 0.5 M NaCl.
Molecular Characterization of Epiphytic Bacterial Communities on Charophycean Green Algae
Authors: Fisher Madeline M. , Wilcox Lee W. , Graham Linda E.
Published: December 2019

Epiphytic bacterial communities within the sheath material of three filamentous green algae,
Desmidium grevillii
,
Hyalotheca dissiliens
, and
Spondylosium pulchrum
(class Charophyceae, order Zygnematales), collected from a
Sphagnum
bog were characterized by PCR amplification, cloning, and sequencing of 16S ribosomal DNA. A total of 20 partial sequences and nine different sequence types were obtained, and one sequence type was recovered from the bacterial communities on all three algae. By phylogenetic analysis, the cloned sequences were placed into several major lineages of the
Bacteria
domain: the
Flexibacter/Cytophaga/Bacteroides
phylum and the α, β, and γ subdivisions of the phylum
Proteobacteria
. Analysis at the subphylum level revealed that the majority of our sequences were not closely affiliated with those of known, cultured taxa, although the estimated evolutionary distances between our sequences and their nearest neighbors were always less than 0.1 (i.e., greater than 90% similar). This result suggests that the majority of sequences obtained in this study represent as yet phenotypically undescribed bacterial species and that the range of bacterial-algal interactions that occur in nature has not yet been fully described.
Comparison of the BAX for Screening/
E. coli
O157:H7 Method with Conventional Methods for Detection of Extremely Low Levels of
Escherichia coli
O157:H7 in Ground Beef
Authors: Johnson Jennifer L. , Brooke Cheryl L. , Fritschel Scott J.
Published: December 2019

Escherichia coli
O157:H7 is an important food-borne pathogen. Often
E. coli
O157:H7 is difficult to detect, because it is present sporadically at very low levels together with very high levels of competitor organisms which can be difficult to distinguish phenotypically. Cultural methods are time-consuming and give variable results in the detection of
E. coli
O157:H7. This study examined the performance of BAX for Screening/
E. coli
O157:H7, a new rapid method for the detection of
E. coli
O157:H7, against traditional and improved cultural methods and an immunodiffusion assay. All cultural methods demonstrated inadequacy in detecting the presence of
E. coli
O157:H7 in inoculated samples. The limitations of these cultural methods further complicate evaluation of screening methodologies. The BAX for Screening/
E. coli
O157:H7 assay outperformed the other methods, with a detection rate of 96.5%, compared to 39% for the best cultural method and 71.5% for the immunodiffusion method. The BAX for Screening/
E. coli
O157:H7 assay proved to be a rapid, highly sensitive test for the detection of low levels of
E. coli
O157:H7 in ground beef.
Immunomagnetic Separation of
Cryptosporidium parvum
from Source Water Samples of Various Turbidities
Authors: Bukhari Z. , McCuin R. M. , Fricker C. R. , Clancy J. L.
Published: December 2019

Immunomagnetic separation (IMS) procedures which specifically capture
Cryptosporidium
oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-
Cryptosporidium
; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of
Cryptosporidium
oocysts.
Analysis of Subfossil Molecular Remains of Purple Sulfur Bacteria in a Lake Sediment
Authors: Coolen Marco J. L. , Overmann Jörg
Published: December 2019

Molecular remains of purple sulfur bacteria (
Chromatiaceae
) were detected in Holocene sediment layers of a meromictic salt lake (Mahoney Lake, British Columbia, Canada). The carotenoid okenone and bacteriophaeophytin
a
were present in sediments up to 11,000 years old. Okenone is specific for only a few species of
Chromatiaceae
, including
Amoebobacter purpureus
, which presently predominates in the chemocline bacterial community of the lake. With a primer set specific for
Chromatiaceae
in combination with denaturing gradient gel electrophoresis, 16S rRNA gene sequences of four different
Chromatiaceae
species were retrieved from different depths of the sediment. One of the sequences, which originated from a 9,100-year-old sample, was 99.2% identical to the 16S rRNA gene sequence of
A. purpureus
ML1 isolated from the chemocline. Employing primers specific for
A. purpureus
ML1 and dot blot hybridization of the PCR products, the detection limit for
A. purpureus
ML1 DNA could be lowered to 0.004% of the total community DNA. With this approach the DNA of the isolate was detected in 7 of 10 sediment layers, indicating that
A. purpureus
ML1 constituted at least a part of the ancient purple sulfur bacterial community. The concentrations of
A. purpureus
DNA and okenone in the sediment were not correlated, and the ratio of DNA to okenone was much lower in the subfossil sediment layers (2.7 · 10
−6
) than in intact cells (1.4). This indicates that degradation rates are significantly higher for genomic DNA than for hydrocarbon cell constituents, even under anoxic conditions and at the very high sulfide concentrations present in Mahoney Lake.
Inactivation of
Escherichia coli
O157:H7 in Apple Juice by Irradiation
Authors: Buchanan R. L. , Edelson S. G. , Snipes K. , Boyd G.
Published: December 2019

Three strains (932, Ent-C9490, and SEA13B88) of
Escherichia coli
O157:H7 were used to determine the effectiveness of low-dose gamma irradiation for eliminating
E. coli
O157:H7 from apple juice or cider and to characterize the effect of inducing pH-dependent, stationary-phase acid resistance on radiation resistance. The strains were grown in tryptic soy broth with or without 1% dextrose for 18 h to produce cells that were or were not induced to pH-dependent stationary-phase acid resistance. The bacteria were then transferred to clarified apple juice and irradiated at 2°C with a cesium-137 irradiator. Non-acid-adapted cells had radiation
D
values (radiation doses needed to decrease a microbial population by 90%) ranging from 0.12 to 0.21 kGy.
D
values increased to 0.22 to 0.31 kGy for acid-adapted cells. When acid-adapted SEA13B88 cells were tested in five apple juice brands having different levels of suspended solids (absorbances ranging from 0.04 to 2.01 at 550 nm), radiation resistance increased with increasing levels of suspended solids, with
D
values ranging from 0.26 to 0.35 kGy. Based on these results, a dose of 1.8 kGy should be sufficient to achieve the 5
D
inactivation of
E. coli
recommended by the National Advisory Committee for Microbiological Criteria for Foods.
Use of an Intelligent Control System To Evaluate Multiparametric Effects on Iron Oxidation by Thermophilic Bacteria
Authors: Stoner Daphne L. , Miller Karen S. , Fife Dee Jay , Larsen Eric D. , Tolle Charles R. , Johnson John A.
Published: December 2019

A learning-based intelligent control system, the BioExpert, was developed and applied to the evaluation of multiparametric effects on iron oxidation by enrichment cultures of moderately thermophilic, acidophilic mining bacteria. The control system acquired and analyzed the data and then selected and maintained the sets of conditions that were evaluated. Through multiple iterations, the BioExpert selected sets of conditions that resulted in improved iron oxidation rates. The results obtained with the BioExpert suggested that temperature and pH were coupled, or interactive, parameters. Elevated temperatures (51.5°C) in combination with a moderately high pH (pH 1.84) impaired the growth of and iron oxidation by the enrichment culture. Moderate-to-high oxidation rates were achieved with a relatively high pH in combination with a relatively low temperature or, conversely, with a relatively low pH in combination with a relatively high temperature. The interactive effect of pH and temperature was not apparent from the results obtained in an experiment in which temperature was the only parameter that was varied. When the BioExpert was applied to a mixed culture containing mesophilic and thermophilic bacteria, the computer “learned” that pH 1.8, 45°C, and an inlet iron concentration from 30 to 35 mM were most favorable for iron oxidation. In conclusion, this study demonstrated that the learning-based intelligent control system BioExpert was an effective experimental tool that can be used to examine multiparametric effects on the growth and metabolic activity of mining bacteria.
Evidence for the Interplay between Trehalose Metabolism and Hsp104 in Yeast
Authors: Iwahashi Hitoshi , Nwaka Solomon , Obuchi Kaoru , Komatsu Yasuhiko
Published: December 2019

Disruption of the
HSP104
gene in a mutant which cannot accumulate trehalose during heat shock treatment caused trehalose accumulation (H. Iwahashi, K. Obuchi, S. Fujii, and Y. Komatsu, Lett. Appl. Microbiol 25:43–47, 1997). This implies that Hsp104 affects trehalose metabolism. Thus, we measured the activities of enzymes involved in trehalose metabolism. The activities of trehalose-synthesizing and -hydrolyzing enzymes are low in the
HSP104
disruption mutant during heat shock. This data is correlated with intracellular trehalose and glucose levels observed in the
HSP104
disruption mutant. These results suggest that during heat shock, Hsp104 contributes to the simultaneous increase in both accumulation and degradation of trehalose.
Heterogeneity among Isolates of
Vibrio vulnificus
Recovered from Eels (
Anguilla anguilla
) in Denmark
Authors: Høi Lise , Dalsgaard Inger , DePaola Angelo , Siebeling Ronald J. , Dalsgaard Anders
Published: December 2019

The findings of this study demonstrate that
Vibrio vulnificus
isolates recovered from diseased eels in Denmark are heterogeneous as shown by O serovars, capsule types, ribotyping, phage typing, and plasmid profiling. The study includes 85
V. vulnificus
isolates isolated from the gills, intestinal contents, mucus, spleen, and kidneys of eels during five disease outbreaks on two Danish eel farms from 1995 to 1997, along with a collection of 12
V. vulnificus
reference strains. The results showed that more than one serovar may be capable of causing disease in eels and that these isolates are genetically heterogenous as shown by ribotyping. Ribotyping also showed that the same isolates may persist in an eel farm and cause recurrent outbreaks. Phage typing did not correlate with ribotyping or serotyping. However, we observed that 26 of 28 isolates, which were not susceptible to any of the phages, showed the same ribotype, O serovar, and capsule type. This suggests that these isolates may possess features that make them resistant to lysis by the phages used in this study. Ninety-three of 97 isolates harbored between one and three high-molecular-weight plasmids which previously had been suggested to be associated with eel virulence. The subdivision of
V. vulnificus
into two biotypes based on the indole reaction can no longer be supported, since 82 of 97 isolates in this study were indole positive, and a subdivision into serovars appears to be more correct.
Effect of Canavanine from Alfalfa Seeds on the Population Biology of
Bacillus cereus
Authors: Emmert Elizabeth A. B. , Milner Jocelyn L. , Lee Julie C. , Pulvermacher Kristie L. , Olivares Heidi A. , Clardy Jon , Handelsman Jo
Published: December 2019

Bacillus cereus
UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685–691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against
B. cereus
,
Bacillus thuringiensis
, and
Vibrio cholerae
. The
B. cereus
UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of
B. cereus.
Cloning and Characterization of NADP-Mannitol Dehydrogenase cDNA from the Button Mushroom,
Agaricus bisporus
, and Its Expression in Response to NaCl Stress
Authors: Stoop Johan M. H. , Mooibroek Hans
Published: December 2019

Mannitol, a six-carbon sugar alcohol, is the main storage carbon in the button mushroom,
Agaricus bisporus
. Given the physiological importance of mannitol metabolism in growth, fruit body development, and salt tolerance of
A. bisporus
, the enzyme responsible for mannitol biosynthesis, NADP-dependent mannitol dehydrogenase (MtDH) (EC
1.1.1.138
), was purified to homogeneity, and
MtDH
cDNA was cloned, sequenced, and characterized. To our knowledge, this represents the first report on the isolation of a cDNA encoding an NADP-dependent mannitol dehydrogenase. The
MtDH
cDNA contains an open reading frame of 789 bp encoding a protein of approximately 28 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified MtDH subunit, whereas the amino acid composition of the deduced protein was nearly identical to that of the purified MtDH. The
MtDH
cDNA showed high homology with a plant-induced short-chain dehydrogenase from
Uromyces fabae
. Phylogenetic analysis based on amino acid sequences from mannitol(-1-phosphate) dehydrogenases indicated a close relationship between the substrate specificity of the enzymes and phylogenetic differentiation. Salt-stressed fruit bodies showed an overall increase in mannitol biosynthesis, as was evident from the increase in MtDH activity, MtDH abundance, and
MtDH
RNA accumulation. Furthermore, the
MtDH
transcript level seems to be under developmental control, as
MtDH
RNA accumulated during maturation of the fruit body.
Insertion-Duplication Mutagenesis in
Streptococcus pneumoniae
: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool
Authors: Lee Myeong S. , Seok Chaok , Morrison Donald A.
Published: December 2019

To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in
Streptococcus pneumoniae
, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.
Use of a Genetically Enhanced, Pediocin-Producing Starter Culture,
Lactococcus lactis
subsp.
lactis
MM217, To Control
Listeria monocytogenes
in Cheddar Cheese
Authors: Buyong Nurliza , Kok Jan , Luchansky John B.
Published: December 2019

Cheddar cheese was prepared with
Lactococcus lactis
subsp.
lactis
MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 10
6
CFU per ml) and a mixture of three
Listeria monocytogenes
strains (ca. 10
3
CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture
L. lactis
subsp.
lactis
MM210, the counts of the pathogen increased to about 10
7
CFU per g after 2 weeks of ripening and then gradually decreased to about 10
3
CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of
L. monocytogenes
decreased to 10
2
CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against
L. monocytogenes
.
Enumeration and Detection of Anaerobic Ferrous Iron-Oxidizing, Nitrate-Reducing Bacteria from Diverse European Sediments
Authors: Straub Kristina L. , Buchholz-Cleven Berit E. E.
Published: December 2019

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 × 10
5
to 5 × 10
8
ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.
Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from
Aspergillus aculeatus
Authors: Murai Toshiyuki , Ueda Mitsuyoshi , Kawaguchi Takashi , Arai Motoo , Tanaka Atsuo
Published: December 2019

Since
Saccharomyces cerevisiae
lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungus
Aspergillus aculeatus
were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into
S. cerevisiae
. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from
Rhizopus oryzae
for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from
S. cerevisiae
. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by
S. cerevisiae
expressing heterologous cellulase genes.
Expression of Active Human Tissue-Type Plasminogen Activator inEscherichia coli
Authors: Qiu Ji , Swartz James R. , Georgiou George
Published: December 2019

The formation of native disulfide bonds in complex eukaryotic proteins expressed inEscherichia coliis extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA inE. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilizedErythrina caffrainhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coliwithout the need for in vitro refolding.
Ralstonia solanacearum
Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence
Authors: Tans-Kersten Julie , Guan Yanfen , Allen Caitilyn
Published: December 2019

Ralstonia
(
Pseudomonas
)
solanacearum
causes bacterial wilt, a serious disease of many crop plants. The pathogen produces several extracellular plant cell wall-degrading enzymes, including polygalacturonases (PGs) and pectin methylesterase (Pme). Pme removes methyl groups from pectin, thereby facilitating subsequent breakdown of this cell wall component by PGs, which are known bacterial wilt virulence factors.
R. solanacearum
PGs could not degrade 93% methylated pectin unless the substrate was first demethylated by Pme, but as the degree of methylation of the pectin substrate decreased, PG activity increased. Primers derived from a published
pme
sequence generated an 800-bp DNA probe fragment, which identified Pme-encoding plasmids from a
R. solanacearum
genomic library. A
pme
chromosomal mutant had no detectable Pme activity in vitro and no longer grew on 93% methylated pectin as a carbon source. Curiously, the
pme
mutant, which had no detectable PG activity on highly methylated pectin, was just as virulent as the wild-type strain on tomato, eggplant (aubergine), and tobacco. Since PG activity is required for full virulence, this result suggests that the pectin in these particular hosts may not be highly methylated, or that the breakdown of highly methylated pectin is not a significant factor in the disease process in general. A positive response regulator of PG production called PehR was not required for wild-type Pme production. However, a mutant strain lacking PhcA, which is a global regulator of several virulence genes, produced no detectable Pme activity. Thus,
pme
expression is directly or indirectly regulated by PhcA but not by PehR.
Utility of Green Fluorescent Nucleic Acid Dyes and Aluminum Oxide Membrane Filters for Rapid Epifluorescence Enumeration of Soil and Sediment Bacteria
Authors: Weinbauer Markus G. , Beckmann Christiane , Höfle Manfred G.
Published: December 2019

High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4′,6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum oxide membrane filters. Due to the bright staining of cells, we recommend SYBR Green II as dye.
Recombinant
Klebsiella oxytoca
Strains with Improved Efficiency in Removal of High Nitrate Loads
Authors: Piñar Guadalupe , Ramos Juan L.
Published: December 2019

Klebsiella oxytoca
CECT 4460 removes high nitrate loads from industrial wastewaters without accumulation of nitrite under optimal culture conditions; however, under nonoptimal conditions nitrite accumulates. This situation reflects an in vivo-limited functioning of nitrite reductase in this strain. As a way to overcome this limitation, an increase in the nitrite reductase gene dose in
K. oxytoca
CECT 4460 was considered. To achieve this, we cloned and transferred into this strain the
Klebsiella pneumoniae nasB
gene, which encodes assimilatory nitrite reductase (Lin et al., J. Bacteriol. 176:2551–2559, 1994). The delivery vector was either the wide-host-range plasmid pUPE2, in which the
nasB
gene is expressed from the
Escherichia coli
P
lac
promoter, or a mini-Tn
5
-Km vector, which upon random insertion in the host chromosome allowed expression of the
nasB
gene from an unidentified chromosomal host promoter. The effect of the increase in the dose of the
nasB
gene in
K. oxytoca
CECT 4460 on the accumulation of nitrite in the culture medium was tested in two recombinant strains. The results obtained showed that
K. oxytoca
CECT 4460 bearing pUPE2 accumulated 88% less nitrite than the wild-type strain, while the recombinant strain bearing the
K. pneumoniae nasB
gene in the host chromosome showed a 25% lower level of nitrite accumulation in the culture medium than that of the wild type.
Occurrence of Male-Specific Bacteriophage in Feral and Domestic Animal Wastes, Human Feces, and Human-Associated Wastewaters
Authors: Calci Kevin R. , Burkhardt William , Watkins William D. , Rippey Scott R.
Published: December 2019

Male-specific bacteriophage (MSB) densities were determined in animal and human fecal wastes to assess their potential impact on aquatic environments. Fecal samples (1,031) from cattle, chickens, dairy cows, dogs, ducks, geese, goats, hogs, horses, seagulls, sheep, and humans as well as 64 sewerage samples were examined for MSB. All animal species were found to harbor MSB, although the great majority excreted these viruses at very low levels. The results from this study demonstrate that in areas affected by both human and animal wastes, wastewater treatment plants are the principal contributors of MSB to fresh, estuarine, and marine waters.
Delineating the Specific Influence of Virus Isoelectric Point and Size on Virus Adsorption and Transport through Sandy Soils
Authors: Dowd Scot E. , Pillai Suresh D. , Wang Sookyun , Corapcioglu M. Yavuz
Published: December 2019

Many of the factors controlling viral transport and survival within the subsurface are still poorly understood. In order to identify the precise influence of viral isoelectric point on viral adsorption onto aquifer sediment material, we employed five different spherical bacteriophages (MS2, PRD1, Qβ, φX174, and PM2) having differing isoelectric points (pI 3.9, 4.2, 5.3, 6.6, and 7.3 respectively) in laboratory viral transport studies. We employed conventional batch flowthrough columns, as well as a novel continuously recirculating column, in these studies. In a 0.78-m batch flowthrough column, the smaller phages (MS2, φX174, and Qβ), which had similar diameters, exhibited maximum effluent concentration/initial concentration values that correlated exactly with their isoelectric points. In the continuously recirculating column, viral adsorption was negatively correlated with the isoelectric points of the viruses. A model of virus migration in the soil columns was created by using a one-dimensional transport model in which kinetic sorption was used. The data suggest that the isoelectric point of a virus is the predetermining factor controlling viral adsorption within aquifers. The data also suggest that when virus particles are more than 60 nm in diameter, viral dimensions become the overriding factor.
Three Phylogenetic Groups of
nodA
and
nifH
Genes in
Sinorhizobium
and
Mesorhizobium
Isolates from Leguminous Trees Growing in Africa and Latin America
Authors: Haukka Kaisa , Lindström Kristina , Young J. Peter W.
Published: December 2019

The diversity and phylogeny of
nodA
and
nifH
genes were studied by using 52 rhizobial isolates from
Acacia senegal
,
Prosopis chilensis
, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera
Sinorhizobium
and
Mesorhizobium
, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the
nodA
and
nifH
genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the
nodA
genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne
nifH
was demonstrated by hybridization for some examples. The
nodA
and
nifH
genes of
Sinorhizobium teranga
ORS1009
T
grouped with the
nodA
and
nifH
genes of the other African sinorhizobia, but
Sinorhizobium saheli
ORS609
T
had a totally different
nodA
sequence, although it was closely related based on the 16S rRNA gene and
nifH
data. This might be because this
S. saheli
strain was originally isolated from
Sesbania
sp., which belongs to a different cross-nodulation group than
Acacia
and
Prosopis
spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.
Effects of High Pressure on Inactivation Kinetics and Events Related to Proton Efflux in
Lactobacillus plantarum
Authors: Wouters Patrick C. , Glaasker Erwin , Smelt Jan P. P. M.
Published: December 2019

Knowledge of the mechanism of pressure-induced inactivation of microorganisms could be helpful in defining an effective, relatively mild pressure treatment as a means of decontamination, especially in combination with other physical treatments or antimicrobial agents. We have studied the effect of high pressure on
Lactobacillus plantarum
grown at pH 5.0 and 7.0. The classical inactivation kinetics were compared with a number of events related to the acid-base physiology of the cell, i.e., activity of F
0
F
1
ATPase, intracellular pH, acid efflux, and intracellular ATP pool. Cells grown at pH 5.0 were more resistant to pressures of 250 MPa than were cells grown at pH 7.0. This difference in resistance may be explained by a higher F
0
F
1
ATPase activity, better ability to maintain a ΔpH, or a higher acid efflux of the cells grown at pH 5.0. After pressure treatment at 250 MPa, the F
0
F
1
ATPase activity was decreased, the ability to maintain a ΔpH was reduced, and the acid efflux was impaired. The ATP pool increased initially after mild pressure treatment and finally decreased after prolonged treatment. The observations on acid efflux and the ATP pool suggest that the glycolysis is affected by high pressure later than is the F
0
F
1
ATPase activity. Although functions related to the membrane-bound ATPase activity were impaired, no morphological changes of the membrane could be observed.
Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family
Authors: Gettemy Jessica M. , Ma Biao , Alic Margaret , Gold Michael H.
Published: December 2019

Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus
Phanerochaete chrysosporium
is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequenced
P. chrysosporium mnp
genes, i.e.,
mnp1
,
mnp2
, and
mnp3
. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 μM Mn, the levels of the
mnp1
and
mnp2
transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of the
mnp3
transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the
mnp1
transcript was present at relatively low levels on days 3 through 7, the
mnp2
transcript level peaked on day 5 and the
mnp3
transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures,
mnp2
was the major expressed
mnp
gene, whereas in large agitated cultures,
mnp1
was the major expressed
mnp
gene.
Prevalence of Broad-Host-Range Lytic Bacteriophages of
Sphaerotilus natans
,
Escherichia coli
, and
Pseudomonas aeruginosa
Authors: Jensen Ellen C. , Schrader Holly S. , Rieland Brenda , Thompson Thomas L. , Lee Kit W. , Nickerson Kenneth W. , Kokjohn Tyler A.
Published: December 2019

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with
Sphaerotilus natans
ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.
Optimization of Exopolysaccharide Production by
Lactobacillus delbrueckii
subsp.
bulgaricus
RR Grown in a Semidefined Medium
Authors: Kimmel Stacy A. , Roberts Robert F. , Ziegler Gregory R.
Published: December 2019

The optimal fermentation temperature, pH, and Bacto-casitone (Difco Laboratories, Detroit, Mich.) concentration for production of exopolysaccharide by
Lactobacillus delbrueckii
subsp.
bulgaricus
RR in a semidefined medium were determined by using response surface methods. The design consisted of 20 experiments, 15 unique combinations, and five replications. All fermentations were conducted in a fermentor with a 2.5-liter working volume and were terminated when 90% of the glucose in the medium had been consumed. The population of
L. delbrueckii
subsp.
bulgaricus
RR and exopolysaccharide content were measured at the end of each fermentation. The optimum temperature, pH, and Bacto-casitone concentration for exopolysaccharide production were 38°C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions was 354 mg of exopolysaccharide/liter, which was within the 95% confidence interval (217 to 374 mg of exopolysaccharide/liter). An additional experiment conducted under optimum conditions showed that exopolysaccharide production was growth associated, with a specific production at the endpoint of 101.4 mg/g of dry cells. Finally, to obtain material for further characterization, a 100-liter fermentation was conducted under optimum conditions. Twenty-nine grams of exopolysaccharide was isolated from centrifuged, ultrafiltered fermentation broth by ethanol precipitation.
Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in
Saccharomyces cerevisiae
Authors: Casal Margarida , Cardoso Helena , Leão Cecília
Published: December 2019

In glucose-grown cells of
Saccharomyces cerevisiae
IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of
V
max
versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of
S. cerevisiae
, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.
Cloning and Nucleotide Sequence of the
gyrB
Gene of
Vibrio parahaemolyticus
and Its Application in Detection of This Pathogen in Shrimp
Authors: Venkateswaran Kasthuri , Dohmoto Nobuhiko , Harayama Shigeaki
Published: December 2019

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of
Vibrio parahaemolyticus
from closely related species, we employed the gyrase B gene (
gyrB
) as a molecular diagnostic probe. The
gyrB
genes of
V. parahaemolyticus
and closely related
Vibrio alginolyticus
were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within
gyrB
specific for
V. parahaemolyticus
. These primers recognized 117 of 117 reference and wild-type
V. parahaemolyticus
strains, whereas amplification did not occur when 90 strains of 37 other
Vibrio
species or 60 strains representing 34 different nonvibrio species were tested. In 100-μl PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of
gyrB
primers for the routine identification of
V. parahaemolyticus
in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp
V. parahaemolyticus
-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of
V. parahaemolyticus
cells per g of shrimp homogenate. By this approach, we were able to detect
V. parahaemolyticus
in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of
V. parahaemolyticus
in shrimp.
Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by
Metarhizium anisopliae
Authors: St. Leger Raymond J. , Joshi Lokesh , Roberts Donald
Published: December 2019

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of
Metarhizium anisopliae
in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.