bluuebunny/crossref_metadata_2025 huggingface dataset:
Dense Community of Hyperthermophilic Sulfur-Dependent Heterotrophs in a Geothermally Heated Shallow Submarine Biotope near Kodakara-Jima Island, Kagoshima, Japan
Authors: Hoaki T , Nishijima M , Miyashita H , Maruyama T
Published: January 2020

Microbial communities in marine hydrothermal sediments (0 to 30 cm deep) in an inlet of Kodakara-Jima Island, Kagoshima, Japan, were studied with reference to environmental factors, especially the presence of amino acids. The study area was shallow, and the sea floor was covered with sand through which hot volcanic gas bubbled and geothermally heated water seeped out. The total bacterial density increased with depth in the sediments in parallel with a rise in the ambient temperature (80(deg)C at the surface and 104(deg)C at a depth of 30 cm in the sediments). As estimated by most-probable-number studies, hyperthermophilic sulfur-dependent heterotrophs growing at 90(deg)C dominated the microbial community (3 x 10(sup7) cells (middot) g of sediment(sup-1) at a depth of 30 cm in the sediments), followed in abundance by hyperthermophilic sulfur-dependent facultative autotrophs (3.3 x 10(sup2) cells (middot) g of sediment(sup-1)). The cooler sandy or rocky floor surrounding the hot spots was covered with white bacterial mats which consisted of large Beggiatoa-like filaments. Both the total organic carbon content, most of which was particulate (75% in the surface sediments), and the amino acid concentration in void seawater in the sediments decreased with depth. Amino acids, both hydrolyzable and free, constituted approximately 23% of the dissolved organic carbon in the surface sediments. These results indicate that a lower amino acid concentration is probably due to consumption by dense populations of hyperthermophilic sulfur-dependent heterotrophs, which require amino acids for their growth and thus create a gradient of amino acid concentration in the sediments. The role of primary producers, which supply essential amino acids to sustain this microbial community, is also discussed.
Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution
Authors: Prijambada I D , Negoro S , Yomo T , Urabe I
Published: January 2020

Through selective cultivation with 6-aminohexanoate linear dimer, a by-product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains.
Purification and characterization of xenorhabdicin, a phage tail-like bacteriocin, from the lysogenic strain F1 of Xenorhabdus nematophilus
Authors: Thaler J O , Baghdiguian S , Boemare N
Published: January 2020

Xenorhabdicin, the phage tail-like bacteriocins of Xenorhabdus nematophilus, and phage head particles, elements produced together after mitomycin induction in X. nematophilus lysogenic strain F1 cultures, were separated by DEAE chromatography, examined by transmission electron microscopy, and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis of xenorhabdicin showed two major subunits of 43 and 20 kDa corresponding to the sheath and the inner core, respectively. At least five other minor subunits of 67, 54, 35, 28, and 16 kDa were also characterized. Electrophoresis of the phage head capsids showed a major 40-kDa subunit and two minor 50- and 34-kDa subunits. Bactericidal activity recorded against closely related bacterial species and spontaneously produced by X. nematophilus resides in the xenorhabdicin particles and is another antimicrobial barrier to save the symbiotic association.
Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1
Authors: Shao Z Q , Behki R
Published: January 2020

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.
Evidence for para dechlorination of polychlorobiphenyls by methanogenic bacteria
Authors: Ye D , Quensen J I , Tiedje J M , Boyd S A
Published: January 2020

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.
Genes Essential for Amber Disease in Grass Grubs Are Located on the Large Plasmid Found in Serratia entomophila and Serratia proteamaculans
Authors: Grkovic S , Glare T R , Jackson T A , Corbett G E
Published: January 2020

The bacteria Serratia entomophila and S. proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Disease symptoms include rapid cessation of feeding and amber coloration of larvae. A 105-kb plasmid (designated pADAP) has consistently been found only in pathogenic isolates of both species. Investigations into the involvement of pADAP in amber disease have been hindered by the lack of both a selectable marker on the plasmid and a reliable transposon delivery system. Kanamycin-resistant transposon insertions into three cloned HindIII fragments (9.5, 9.6, and 10.6 kb) were isolated and introduced into pADAP by shuttle mutagenesis. Inserts into the 9.5-and 9.6-kb HindIII fragments on pADAP did not alter disease-causing ability. When plasmids with inserts into the 9.6-kb region were conjugated into plasmid-minus, nonpathogenic isolates of S. entomophila and S. proteamaculans, all of them became pathogenic. Transposon insertions into two regions of the 10.6-kb HindIII fragment continued to cause cessation of feeding but failed to produce amber coloration. Further analysis of a mutant from each amber-minus region (pADK-10 and pADK-13) demonstrated that the antifeeding effect was produced only at dosages higher than that of the wild-type strain. Complementation with the wild-type HindIII fragment restored full-blown disease properties for pADK-13, but not for pADK-10.
Viral tracer studies indicate contamination of marine waters by sewage disposal practices in key largo, Florida
Authors: Paul J H , Rose J B , Brown J , Shinn E A , Miller S , Farrah S R
Published: January 2020

Domestic wastewater disposal practices in the Florida Keys are primarily limited to on-site disposal systems such as septic tanks, injection wells, and illegal cesspits. Poorly treated sewage is thus released into the highly porous subsurface Key Largo limestone matrix. To investigate the fate and transport of sewage in the subsurface environment and the potential for contamination of marine surface waters, we employed bacteriophages as tracers in a domestic septic system and a simulated injection well in Key Largo, Florida. Transport of bacteriophage (Phi)HSIC-1 from the septic tank to adjacent surface canal waters and outstanding marine waters occurred in as little as 11 and 23 h, respectively. Transport of the Salmonella phage PRD1 from the simulated injection well to a canal adjacent to the injection site occurred in 11.2 h. Estimated rates of migration of viral tracers ranged from 0.57 to 24.2 m/h, over 500-fold greater than flow rates measured previously by subsurface flow meters in similar environments. These results suggest that current on-site disposal practices can lead to contamination of the subsurface and surface marine waters in the Keys.
Massive fungal contamination in animal care facilities traced to bedding supply
Authors: Mayeux P , Dupepe L , Dunn K , Balsamo J , Domer J
Published: January 2020

During the course of immunologic studies involving the gastrointestinal colonization of mice with Candida albicans, it became apparent that the animals were being exposed to large numbers of Aspergillus fumigatus spores which interfered with the C. albicans colonization. To determine the source of the A. fumigatus exposure and the extent of fungal contamination of the medical school vivarium and four satellite facilities, fungal analyses of feed, bedding, and air were undertaken. Initial samples from the air were collected with 3-h settle plates; air sampling following cleanup was done with an Anderson air sampler. The source of contamination in the mouse rooms was determined to be Beta Chip bedding, which came from the manufacturer highly contaminated. Beta Chip bedding (1 g) obtained from the manufacturer just prior to testing contained 10(4) CFU of A. fumigatus, 20 CFU of a zygomycete, and 10 CFU of a Penicillium sp. Coarse-grade Beta Chip had approximately one-half those levels of contamination. Pure Cob bedding was highly contaminated also, but with a Fusarium sp. and a Cladosporium sp. Untreated and heat-treated Sani-Chip as well as all other heat-treated preparations obtained from the manufacturer contained no detectable spores. Rodent chow direct from the manufacturer had no A. fumigatus, although it did contain 150 CFU of fungus per g, including 80 CFU of a Rhodotorula sp., 60 CFU of Cryptococcus uniguttulatus, and 1 CFU of a Penicillium sp.(ABSTRACT TRUNCATED AT 250 WORDS)
Host-Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strains in Serogroup 110
Authors: Lohrke S M , Orf J H , Martinez-Romero E , Sadowsky M J
Published: January 2020

We previously reported the identification of a soybean plant introduction (PI) genotype, PI 417566, which restricts nodulation by Bradyrhizobium japonicum MN1-1c (USDA 430), strains in serogroup 129, and USDA 110 (P. B. Cregan, H. H. Keyser, and M. J. Sadowsky, Appl. Environ. Microbiol. 55:2532-2536, 1989, and Crop Sci. 29:307-312, 1989). In this study, we further characterized nodulation restriction by PI 417566. Twenty-four serogroup 110 isolates were tested for restricted nodulation on PI 417566. Of the 24 strains examined, 62.5% were restricted in nodulation by the PI genotype. The remainder of the serogroup 110 strains tested (37.5%), however, formed significant numbers of nodules on PI 417566, suggesting that host-controlled restriction of nodulation by members of serogroup 110 is strain dependent. Analysis of allelic variation at seven enzyme-encoding loci by multilocus enzyme electrophoresis indicated that the serogroup 110 isolates can be divided into two major groups. The majority of serogroup 110 isolates which nodulated PI 417566 belonged to the same multilocus enzyme electrophoresis group. B. japonicum USDA 110 and USDA 123 were used as coinoculants in competition-for-nodulation studies using PI 417566. Over 98% of the nodules formed on PI 417566 contained USDA 123, whereas less than 2% contained USDA 110. We also report the isolation of a Tn5 mutant of USDA 110 which has overcome nodulation restriction conditioned by PI 417566. This mutant, D4.2-5, contained a single Tn5 insertion and nodulated PI 417566 to an extent equal to that seen with the unrestricted strain USDA 123. The host range of D4.2-5 on soybean plants and other legumes was unchanged relative to that of USDA 110, except that the mutant nodulated Glycine max cv. Hill more efficiently. While strain USDA 110 has the ability to block nodulation by D4.2-5 on PI 417566, the nodulation-blocking phenomenon was not seen unless strain USDA 110 was inoculated at a 100-fold greater concentration than the mutant strain.
Enhanced fermentation of mannitol and release of cytotoxin by Clostridium difficile in alkaline culture media
Authors: Kazamias M T , Sperry J F
Published: January 2020

Clostridium difficile ATCC 43255 fermented less than 10% of the mannitol in a medium at pH 7; however, when the initial pH of the medium was adjusted to 8.5 or 9, about 80% of the mannitol was fermented. Cell extracts of C. difficile phosphorylated mannitol with phosphoenolpyruvate, not ATP, indicating a phosphoenolpyruvate phosphotransferase system transport phosphorylation of mannitol. The phosphorylation product was dehydrogenated by D-mannitol-1-phosphate:NAD oxidoreductase. Growth at an initial pH of 8.5 yielded cytotoxin titers of 10(7) to 10(8) in Trypticase-yeast extract-mannitol medium, wit a titer of 10(8) as early as 13 h.
Development of a monoclonal antibody-based immunoassay for the detection and quantification of Anguillospora longissima colonizing leaf material
Authors: Bermingham S , Dewey F M , Maltby L
Published: January 2020

Monoclonal antibodies (MAbs) to the aquatic hyphomycete Anguillospora longissima were raised in mice by using a coimmunization program. A cell line was raised that produced a MAb of the immunoglobulin M class that was specific for A. longissima both in an enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence but that did not recognize other members of the aquatic hyphomycetes. This MAb (AL-HH8c) was used to develop a quantitative ELISA in vitro. The antigen recognized by AL-HH8c is produced throughout the mycelium, irrespective of mycelial age and culture conditions. By using this MAb, mycelium of A. longissima colonizing leaf material can be detected by ELISA, immunofluorescence, and immunoenzymatic staining methods.
A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water
Authors: Pyle B H , Broadaway S C , McFeters G A
Published: January 2020

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.
Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA
Authors: Bereswill S , Bugert P , Bruchm�ller I , Geider K
Published: January 2020

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.
Methylmercury oxidative degradation potentials in contaminated and pristine sediments of the carson river, nevada
Authors: Oremland R S , Miller L G , Dowdle P , Connell T , Barkay T
Published: January 2020

Sediments from mercury-contaminated and uncontaminated reaches of the Carson River, Nevada, were assayed for sulfate reduction, methanogenesis, denitrification, and monomethylmercury (MeHg) degradation. Demethylation of [(sup14)C]MeHg was detected at all sites as indicated by the formation of (sup14)CO(inf2) and (sup14)CH(inf4). Oxidative demethylation was indicated by the formation of (sup14)CO(inf2) and was present at significant levels in all samples. Oxidized/reduced demethylation product ratios (i.e., (sup14)CO(inf2)/(sup14)CH(inf4) ratios) generally ranged from 4.0 in surface layers to as low as 0.5 at depth. Production of (sup14)CO(inf2) was most pronounced at sediment surfaces which were zones of active denitrification and sulfate reduction but was also significant within zones of methanogenesis. In a core taken from an uncontaminated site having a high proportion of oxidized, coarse-grain sediments, sulfate reduction and methanogenic activity levels were very low and (sup14)CO(inf2) accounted for 98% of the product formed from [(sup14)C]MeHg. There was no apparent relationship between the degree of mercury contamination of the sediments and the occurrence of oxidative demethylation. However, sediments from Fort Churchill, the most contaminated site, were most active in terms of demethylation potentials. Inhibition of sulfate reduction with molybdate resulted in significantly depressed oxidized/reduced demethylation product ratios, but overall demethylation rates of inhibited and uninhibited samples were comparable. Addition of sulfate to sediment slurries stimulated production of (sup14)CO(inf2) from [(sup14)C]MeHg, while 2-bromoethanesulfonic acid blocked production of (sup14)CH(inf4). These results reveal the importance of sulfate-reducing and methanogenic bacteria in oxidative demethylation of MeHg in anoxic environments.
Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species
Authors: Farrelly V , Rainey F A , Stackebrandt E
Published: January 2020

In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification. The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T. thermophilus." The values for the pair of Bacillus subtilis and "T. thermophilus" showed greater deviations from the predicted value. The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present.
PCR detection of the lactocin S structural gene in bacteriocin-producing lactobacilli from meat
Authors: Rodr�guez J M , Cintas L M , Casaus P , Su�rez A , Hern�ndez P E
Published: January 2020

The lactocin S structural gene (lasA) in seven bacteriocinogenic lactobacilli isolated from fermented sausages was studied. Two degenerate primers were synthesized to amplify a 75-bp fragment of the gene. Three strains amplified the fragment from their plasmid DNA, and hybridization analysis confirmed these results.
Two-Dimensional Gel Electrophoresis Analysis of the Response of Pseudomonas putida KT2442 to 2-Chlorophenol
Authors: Lupi C G , Colangelo T , Mason C A
Published: January 2020

The effects of exposure of Pseudomonas putida KT2442 to 2-chlorophenol as a model for the chemical stress response were examined by two-dimensional polyacrylamide gel electrophoresis. Individual protein concentrations were determined at 45, 65, and 95 min following the addition of 2-chlorophenol at a concentration of 1.63 mM to exponentially growing cultures of P. putida KT2442 by silver staining the separated proteins. The changes in the protein concentrations could be classified into four categories, namely those which increased continuously during exposure, those which decreased in concentration, those which showed a concentration peak at some point following exposure, and those which were essentially unaffected. Thirty proteins with isoelectric points between pH 4 and 6 increased in concentration, 27 decreased, and 90 had a concentration maximum or minimum between 45 and 95 min. Of those proteins with isoelectric points between 5.5 and 10, 68 increased in concentration, 39 decreased in concentration, and 47 showed a concentration peak in the middle of the sampling period. Thus, in the evaluation of the stress response, a functional description requires an understanding both of proteins which are required at higher concentrations and of those whose presence appears to be no longer essential.
Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi
Authors: Semimaru T , Goto M , Furukawa K , Hayashida S
Published: January 2020

Glucoamylase I (GAI) from Aspergillus awamori var. kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615). In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I. All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3H alpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA delta 470-507, lacking A-470 to S-507, and GA delta GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells. The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI. The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI.(ABSTRACT TRUNCATED AT 250 WORDS)
Abundance of Tn3, Tn21, and Tn501 transposase (tnpA) sequences in bacterial community DNA from marine environments
Authors: Dahlberg C , Hermansson M
Published: January 2020

The occurrence of the tnpA genes of the transposons Tn3, Tn21, and Tn501 was assessed in total bacterial community DNA isolated from different marine environments. The PCR technique was employed, together with most probable number statistics, to determine the abundance of the target tnpA genes. All three genes could be detected, and the Tn21 tnpA sequences predominated in all samples. The smallest amount of total community DNA in which the Tn21 tnpA sequence could be detected was 0.037 ng, and on the basis of our results, we estimated that this sequence was present in 1 of 1,000 to 10,000 bacteria. Hybridization of the PCR products with the respective tnpA probes verified the Tn21 and Tn501 tnpA sequences but only some of the Tn3 tnpA amplification products. The distribution and dissemination of transposons in natural bacterial communities are discussed.
Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66
Authors: Butler M J , Binnie C , DiZonno M A , Krygsman P , Soltes G A , Soostmeyer G , Walczyk E , Malek L T
Published: January 2020

The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.
Suitability of selective plating media for recovering heat- or freeze-stressed Escherichia coli O157:H7 from tryptic soy broth and ground beef
Authors: Rocelle M , Clavero S , Beuchat L R
Published: January 2020

The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7.
Genotyping of rotaviruses isolated from sewage
Authors: Gajardo R , Bouchriti N , Pintó R M , Bosch A
Published: January 2020

Rotaviruses from environmental samples have been genotyped by a seminested reverse transcription PCR assay with serotype-specific primers derived from variable regions of gene 9, which produce different characteristic segment sizes for serotypes 1 to 4. The method enabled the detection and identification of type 1, 2, and 3 group A rotaviruses in sewage.
Production and characterization of monoclonal antibodies against the O-5 antigen of Salmonella typhimurium lipopolysaccharide
Authors: Jaradat Z W , Zawistowski J
Published: January 2020

Three murine monoclonal antibodies (MAbs) were produced by fusion of P3X63-Ag8.653 myeloma cells and splenocytes of a mouse immunized with heat-attenuated (20 min, 80 degrees C) Salmonella typhimurium cells. MAbs 5A5 and 5B2 were of the immunoglobulin M (IgM) class, while MAb 4A8 was IgG2a. All possessed the kappa light chains. The MAbs were specific to the lipopolysaccharide (LPS) O-5 antigen of Salmonella B serogroup, as determined by electrophoresis followed by immunoblotting. All MAbs recognized the same epitope, as determined by an additive enzyme-linked immunosorbent assay (ELISA), although IgM MAbs exhibited higher avidity than the IgG MAb. ELISA analyses revealed that all three MAbs reacted with S. typhimurium (LPS O:1, 4, 5, and 12) while failing to recognize S. typhimurium var. copenhagen (LPS O:1, 4, and 12). The MAbs reacted equally with live and heat-attenuated Salmonella B serovars containing LPS O-5 antigen. The ability of the MAbs to detect live bacterial cells was further confirmed by transmission electron microscopy. Treatment of bacteria with cholic acid and extremely low pH did not affect antibody binding to S. typhimurium. However, when S. typhimurium cells were exposed to alkaline conditions prior to reaction with all three MAbs, no binding was observed. The use of MAbs to discriminate between S. typhimurium and S. typhimurium var. copenhagen in meat samples was investigated.
Purification and characterization of two arabinofuranosidases from solid-state cultures of the fungus Penicillium capsulatum
Authors: Filho E X , Puls J , Coughlan M P
Published: January 2020

Two arabinofuranosidases, termed Ara I and Ara II, from solid-state cultures of Penicillium capsulatum were purified to apparent homogeneity as judged by electrophoresis and isoelectric focusing. Each enzyme is a single subunit glycoprotein, and they have M(r)s and pIs of 64,500 and 4.15 (Ara I) and 62,700 and 4.54 (Ara II), respectively. Ara I is most active at pH 4.0 and 60 degrees C, while Ara II exhibits optimal activity at pH 4.0 and 55 degrees C. Ara I is the more thermostable, with its half-life at 70 degrees C and pH 4.0 being 17.5 min. By contrast, the half-life of Ara II is only 9 min at 60 degrees C and pH 4.0. Ara I has the lower Km and higher catalytic constant values with p-nitrophenyl-alpha-L-arabinofuranoside being used as the substrate. Arabinose, a competitive inhibitor (Ki, 16.4 mM) of Ara II, has no effect on Ara I activity at concentrations of up to 40 mM. Each enzyme catalyzes the release of arabinose from pectin, araban, and certain arabinose-containing xylans. The last activity is enhanced by pretreatment of the relevant substrates with xylanase, ferulic acid esterase, or combinations of these enzymes. Thus, arabinoxylooligosaccharides in which arabinose is the sole side chain substituent appear to be the preferred substrates. On the basis of the evidence cited above, each enzyme has been classified as an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.79).
Enumeration of viable bacteria in the marine pelagic environment
Authors: Bianchi A , Giuliano L
Published: January 2020

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.
Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental Protection Agency-approved membrane filter method
Authors: Brenner K P , Rankin C C , Sivaganesan M , Scarpino P V
Published: January 2020

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium.
Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR
Authors: Atmar R L , Neill F H , Woodley C M , Manger R , Fout G S , Burkhardt W , Leja L , McGovern E R , Le Guyader F , Metcalf T G , Estes M K
Published: January 2020

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.
Importance of Preferential Flow and Soil Management in Vertical Transport of a Biocontrol Strain of Pseudomonas fluorescens in Structured Field Soil
Authors: Natsch A , Keel C , Troxler J , Zala M , Von Albertini N , Defago G
Published: January 2020

The large-scale release of wild-type or genetically modified bacteria into the environment for control of plant diseases or for bioremediation entails the potential risk of groundwater contamination by these microorganisms. For a model study on patterns of vertical transport of bacteria under field conditions, the biocontrol strain Pseudomonas fluorescens CHA0, marked with a spontaneous resistance to rifampin (CHA0-Rif), was applied to a grass-clover ley plot (rotation grassland) and a wheat plot. Immediately after bacterial application, heavy precipitation was simulated by sprinkling, over a period of 8 h, 40 mm of water containing the mobile tracer potassium bromide and the dye Brilliant Blue FCF to identify channels of preferential flow. One day later, a 150-cm-deep soil trench was dug and soil profiles were prepared. Soil samples were extracted at different depths of the profiles and analyzed for the number of CHA0-Rif cells and the concentration of bromide and Brilliant Blue FCF. Dye coverage in the soil profiles was estimated by image analysis. CHA0 was present at 10(sup8) CFU/g in the surface soil, and 10(sup6) to 10(sup7) CFU/g of CHA0 was detected along macropores between 10 and 150 cm deep. Similarly, the concentration of the tracer bromide along the macropores remained at the same level below 20 cm deep. Dye coverage in lower soil layers was higher in the ley than in the wheat plot. In nonstained parts of the profiles, the number of CHA0-Rif cells was substantially smaller and the bromide concentration was below the detection limit in most samples. We conclude that after heavy rainfall, released bacteria are rapidly transported in large numbers through the channels of preferential flow to deeper soil layers. Under these conditions, the transport of CHA0-Rif is similar to that of the conservative tracer bromide and is affected by cultural practice.
Manganese-enhanced biotransformation of atrazine by the white rot fungus Pleurotus pulmonarius and its correlation with oxidation activity
Authors: Masaphy S , Henis Y , Levanon D
Published: January 2020

Manganese enhanced atrazine transformation by the fungus Pleurotus pulmonarius when added to a liquid culture medium at concentrations of up to 300 microM. Both N-dealkylated and propylhydroxylated metabolites accumulated in the culture medium, with the former accumulating to a greater extent than did the latter. Lipid peroxidation, oxygenase and peroxidase activities, and the cytochrome P-450 concentration increased. In addition, an increase in the spectral interactions between atrazine and components in the cell extract was observed. Antioxidants, mainly nordihydroguaiaretic acid, which inhibits lipoxygenase, peroxidase, and P-450 activities, and piperonyl butoxide, which inhibits P-450 activity, inhibited atrazine transformation by the mycelium. It is suggested that the stimulation of oxidative activity by Mn might be responsible for increasing the biotransformation of atrazine and for nonspecific transformations of other xenobiotic compounds.
A simulation of microbial competition in the human colonic ecosystem
Authors: Coleman M E , Dreesen D W , Wiegert R G
Published: January 2020

Many investigations of the interactions of microbial competitors in the gastrointestinal tract used continuous-flow anaerobic cultures. The simulation reported here was a deterministic 11-compartment model coded by using the C programming language and based on parameters from published in vitro studies and assumptions were data were unavailable. The resource compartments were glucose, lactose and sucrose, starch, sorbose, and serine. Six microbial competitors included indigenous nonpathogenic colonizers of the human gastrointestinal tract (Escherichia coli, Enterobacter aerogenes, Bacteroids ovatus, Fusobacterium varium, and Enterococcus faecalis) and the potential human enteropathogen Salmonella typhimurium. Flows of carbon from the resources to the microbes were modified by resource and space controls. Partitioning of resources to the competitors that could utilize them was calculated at each iteration on the basis of availability of all resources by feeding preference functions. Resources did not accumulate during iterations of the model. The results of the computer simulation of microbial competition model and for various modifications of the model. The results were based on few measured parameters but may be useful in the design of user-friendly software to aid researchers in defining and manipulating the microbial ecology of colonic ecosystems as relates to food-borne disease.
Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences
Authors: Du C , Nickerson K W
Published: January 2020

Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis.
Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola)
Authors: Huss M J , Campbell C L , Jennings D B , Leslie J F
Published: January 2020

Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven biological species. While emphasizing the discreteness of the mating populations as biological entities, our isozyme data also reaffirm the close genetic relationship among these groups.
Transposon mutagenesis in Desulfovibrio desulfuricans: development of a random mutagenesis tool from Tn7
Authors: Wall J D , Murnan T , Argyle J , English R S , Rapp-Giles B J
Published: January 2020

The transposons Tn5, Tn7, Tn9, and Tn10 or their derivatives have been examined for transposition in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20. Tn7 inserted with a frequency of 10(-4) to 10(-3) into a unique attachment site that shows strong homology with those sites identified in other gram-negative bacteria. Inactivation of the tnsD gene in Tn7, encoding the function directing insertion into the unique site, yielded a derivative that transposed essentially randomly with a frequency of ca. 10(-6) per donor. Derivatives of Tn5, but not wild-type Tn5, were also found to undergo random transposition at a similar frequency. No evidence was obtained for transposition of Tn9 or Tn10.
The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth
Authors: Wen Z , Morrison M
Published: January 2020

Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria.