bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations Affecting Hyphal Colonization and Pyoverdine Production in Pseudomonads Antagonistic toward
Phytophthora parasitica
Authors: Yang Ching-Hong , Menge John A. , Cooksey Donald A.
Published: January 2020

In previous studies,
Pseudomonas putida
06909 and
Pseudomonas fluorescens
09906 suppressed populations of
Phytophthora parasitica
in the citrus rhizosphere, suggesting that these bacteria may be useful in biological control of citrus root rot. In this study we investigated the mechanisms of antagonism between the bacteria and the fungus. Both bacteria colonized
Phytophthora
hyphae and inhibited the fungus on agar media. A hyphal column assay was developed to measure the colonization of bacteria on fungal hyphae and to enrich for colonization-deficient mutants. In this way we identified Tn
5
mutants of each pseudomonad that were not able to colonize the hyphae and inhibit fungal growth in vitro. Colonization-deficient mutants were nonmotile and lacked flagella. Survival of nonmotile mutants in a citrus soil was similar to survival of a random Tn
5
mutant over a 52-day period. Additional screening of random Tn
5
mutants of both pseudomonads for loss of fungal inhibition in vitro yielded two distinct types of mutants. Mutants of the first type were deficient in production of pyoverdines and in inhibition of the fungus in vitro, although they still colonized fungal hyphae. Mutants of the second type lacked flagella and were not able to colonize the hyphae or inhibit fungal growth. No role was found for antibiotic production by the two bacteria in the inhibition of the fungus. Our results suggest that both hyphal colonization and pyoverdine production are important in the inhibition of
Phytophthora parasitica
by
P. fluorescens
and
P. putida
in vitro.
Production of Chitinases and β-1,3-Glucanases by
Stachybotrys elegans
, a Mycoparasite of
Rhizoctonia solani
Authors: Tweddell Russell J. , Jabaji-Hare Suha H. , Charest Pierre M.
Published: January 2020

The in vitro production of chitinases and β-1,3-glucanases by
Stachybotrys elegans
, a mycoparasite of
Rhizoctonia solani
, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO
3
. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected.
S. elegans
culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading
R. solani
mycelium.
Classification of the uptake hydrogenase-positive (Hup+) bean rhizobia as Rhizobium tropici
Authors: van Berkum P , Navarro R B , Vargas A A
Published: January 2020

Phenotypic and genetic characterization indicated that Hup+ bean rhizobial strains are type IIA and type IIB Rhizobium tropici. The Hup+ strain USDA 2840, which did not cluster with either of the two types of R. tropici in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences similar to those of three reference strains of R. tropici type IIA. The Hup+ strain USDA 2738, which clustered with the reference strain of R. tropici IIB in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences more closely resembling those of the reference strains of R. tropici type IIA than those of type IIB. DNA amplification with the Y1 and Y2 primers to generate a portion of the 16S rDNA operon was useful to distinguish R. tropici type IIA strains from other bean rhizobial strains. The phylogenetic position of the type IIA strain of R. tropici USDA 2840, determined from the partial 16S rDNA sequence, indicated a more distant relationship with the type IIB strain of R. tropici CIAT899 than with the as yet unnamed rhizobial species of Leucaena leucocephala, TAL 1145. Therefore, we suggest that it may be appropriate either to separate R. tropici types IIA and IIB into two different species or to identify TAL 1145 to the species level as a third type of R. tropici.
Secretion of Ligninolytic Enzymes and Mineralization of
14
C-Ring-Labelled Synthetic Lignin by Three
Phlebia tremellosa
Strains
Authors: Vares Tamara , Niemenmaa Outi , Hatakka Annele
Published: January 2020

Production of ligninolytic enzymes by three strains of the white rot fungus
Phlebia tremellosa
(syn.
Merulius tremellosus
) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of
P. tremellosa.
In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 μM) or elevated (24 and 120 μM) Mn(II) concentrations. However, H
2
O
2
- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of
14
C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for
Phanerochaete chrysosporium
and
Phlebia radiata.
A high manganese concentration repressed the evolution of
14
CO
2
even when a chelating agent, sodium malonate, was included in the medium.
Xylose and Glucose Utilization by
Bacteroides xylanolyticus
X5-1 Cells Grown in Batch and Continuous Culture
Authors: Biesterveld Steef , Oude Elferink Stefanie J. W. H. , Zehnder Alexander J. B. , Stams Alfons J. M.
Published: January 2020

During cultivation on a mixture of xylose and glucose,
Bacteroides xylanolyticus
X5-1 showed neither diauxic growth nor a substrate preference. Xylose-limited continuous-culture cells were able to consume xylose and glucose both as single substrates and as mixed substrates without any lag phase. When glucose was the growth-limiting substrate, the microorganism was unable to consume xylose. However, in the presence of a small amount of glucose or pyruvate, xylose was utilized after a short lag phase. In glucose-limited cells, xylose isomerase was present at low activity but xylulose kinase activity could not be detected. On addition of a mixture of xylose and glucose, xylose isomerase was induced immediately and xylulose kinase was induced after about 30 min. The induction of the two enzymes was sensitive to chloramphenicol, showing de novo synthesis. Xylose uptake in glucose-grown cells was very low, but the uptake rate could be increased when incubated with a xylose-glucose mixture. The increase in the uptake rate was not affected by chloramphenicol, indicating that a constitutive uptake system had to be activated. The inability of
B. xylanolyticus
X5-1 cells undergoing glucose-limited continuous culture to induce the xylose catabolic pathway after the addition of only xylose probably was caused by energy limitation.
Purification and Characterization of an Inducible
s
-Triazine Hydrolase from
Rhodococcus corallinus
NRRL B-15444R
Authors: Mulbry Walter W.
Published: January 2020

The widespread use and relative persistence of
s
-triazine compounds such as atrazine and simazine have led to increasing concern about environmental contamination by these compounds. Few microbial isolates capable of transforming substituted
s
-triazines have been identified.
Rhodococcus corallinus
NRRL B-15444 has previously been shown to possess a hydrolase activity that is responsible for the dechlorination of the triazine compounds deethylsimazine (6-chloro-
N
-ethyl-1,3,5-triazine-2,4-diamine) (CEAT) and deethylatrazine (6-chloro-
N
-isopropyl-1,3,5-triazine-2,4-diamine) (CIAT). The enzyme responsible for this activity was purified and shown to be composed of four identical subunits of 54,000 Da. Kinetic experiments revealed that the purified enzyme is also capable of deaminating the structurally related
s
-triazine compounds melamine (2,4,6-triamino-1,3,5-triazine) (AAAT) and CAAT (2-chloro-4,6-diamino-1,3,5-triazine), as well as the pyrimidine compounds 2,4,6-triaminopyrimidine (AAAP) and 4-chloro-2,6-diaminopyrimidine (CAAP). The triazine herbicides atrazine and simazine inhibit the hydrolytic activities of the enzyme but are not substrates. Induction experiments demonstrate that triazine hydrolytic activity is inducible and that this activity rises approximately 20-fold during induction.
Phylogenetic analysis and development of probes for differentiating methylotrophic bacteria
Authors: Brusseau G A , Bulygina E S , Hanson R S
Published: January 2020

Fifteen small-subunit rRNAs from methylotrophic bacteria have been sequenced. Comparisons of these sequences with 22 previously published sequences further defined the phylogenetic relationships among these bacteria and illustrated the agreement between phylogeny and physiological characteristics of the bacteria. Phylogenetic trees were constructed with 16S rRNA sequences from methylotrophic bacteria and representative organisms from subdivisions within the class Proteobacteria on the basis of sequence similarities by using a weighted least-mean-square difference method. The methylotrophs have been separated into coherent clusters in which bacteria shared physiological characteristics. The clusters distinguished bacteria which used either the ribulose monophosphate or serine pathway for carbon assimilation. In addition, methanotrophs and methylotrophs which do not utilize methane were found to form distinct clusters within these groups. Five new deoxyoligonucleotide probes were designed, synthesized, labelled with digoxigenin-11-ddUTP, and tested for the ability to hybridize to RNA extracted from the bacteria represented in the unique clusters and for the ability to detect RNAs purified from soils enriched for methanotrophs by exposure to a methane-air atmosphere for one month. The 16S rRNA purified from soil hybridized to the probe which was complementary to sequences present in 16S rRNA from serine pathway methanotrophs and hybridized to a lesser extent with a probe complementary to sequences in 16S rRNAs of ribulose monophosphate pathway methanotrophs. The nonradioactive detection system used performed reliably at amounts of RNA from pure cultures as small as 10 ng.
Detection of alcohol-tolerant hiochi bacteria by PCR
Authors: Nakagawa T , Shimada M , Mukai H , Asada K , Kato I , Fujino K , Sato T
Published: January 2020

We report a sensitive and rapid method for detection of hiochi bacteria by PCR. This method involves the electrophoresis of amplified DNA. Nucleotide sequences of the spacer region between 16S and 23S rRNA genes of 11 Lactobacillus strains were identified by analysis of PCR products. Five primers were designed by analysis of similarities among these sequences. A single cell of Lactobacillus casei subsp. casei could be detected when purified genomic DNA was used as the template. When various cell concentrations of L. casei subsp. casei were added to 50 ml of pasteurized sake and the cells were recovered, the detection limit was about one cell. No discrete band was observed in electrophoresis after PCR when human, Escherichia coli, mycoplasma, Acholeplasma, yeast, or mold DNA was used as the template.
Patterns of Reactivity between a Panel of Monoclonal Antibodies and Forage
Rhizobium
Strains
Authors: Olsen Perry , Wright Sara , Collins Mandy , Rice Wendell
Published: January 2020

A panel of 11 monoclonal antibodies raised against vegetative cells of
Rhizobium leguminosarum
biovar trifolii or
Rhizobium meliloti
was tested by enzyme-linked immunosorbent assay for reactivity with 47 strains of
R. leguminosarum
biovar trifolii and 60 strains of
R. meliloti.
The goal of the study was to define the degree of specificity associated with each antibody and to gain an understanding of the amount of antigenic diversity found among the strains and between the species. Each antibody was tested against each
Rhizobium
strain in four forms: washed steamed cells, washed unsteamed cells, cell-free culture broth, and nodule squash material. Each antibody showed a different pattern of reactivity among the 107 strains. One of each of the antibodies developed against
R. meliloti
and
R. leguminosarum
biovar trifolii reacted in a highly specific manner with cells or antigen from the immunogenic strain only. Nine of the antibodies recognized secreted as well as cellular antigen from many of the strains. Analysis of patterns of reactivity between the 107 strains and the 11 antibodies separated the strains into 28 groups of which 12 were represented by one strain only.
The alcohol dehydrogenase gene adh1 is induced in Aspergillus flavus grown on medium conducive to aflatoxin biosynthesis
Authors: Woloshuk C P , Payne G A
Published: January 2020

An Aspergillus flavus cDNA library was screened by differential hybridization to isolate clones corresponding to genes that are actively transcribed under culture conditions conducive to aflatoxin biosynthesis. One clone with a 1.28-kb insert was isolated, and its nucleotide sequence was determined. The nucleotide sequence of this clone had 75% DNA identity to those of the alcohol dehydrogenase genes from Aspergillus nidulans, and the putative polypeptide translated from the cDNA sequence had 82% similarity with the amino acid sequences of the A. nidulans proteins. Thus, this gene has been designated adh1. Southern hybridization analysis of genomic DNA from A. flavus indicated that there was one copy of the adh1 gene. Northern (RNA) hybridization analysis indicated that the adh1 transcript accumulated in culture medium conducive to aflatoxin production and the timing of accumulation of adh1 transcripts was similar to that for aflatoxin. Fusion of the promoter region of adh1 to a beta-glucuronidase reporter gene indicated that accumulation of the adh1 transcript was the result of transcriptional activation. These molecular data support previous physiological evidence that showed the importance of carbohydrate metabolism during aflatoxin biosynthesis.
Recombination of the
bph
(Biphenyl) Catabolic Genes from Plasmid pWW100 and Their Deletion during Growth on Benzoate
Authors: Lloyd-Jones Gareth , de Jong Caroline , Ogden Richard C. , Duetz Wouter A. , Williams Peter A.
Published: January 2020

Pseudomonas
sp. strain CB406 was isolated from polychlorinated biphenyl-contaminated soil and harbors a nontransmissible plasmid, pWW100, of approximately 200 kb which carries the genes required for biphenyl and 4-chlorobiphenyl catabolism. The catabolic phenotype was mobilized following the construction in vivo of a cointegrate plasmid containing functional upper and lower biphenyl operons inserted into the broad-host-range R plasmid RP4. The Bph
+
phenotype carried by pWW100 was stable in nonselective media but was unstable during growth on benzoate, where the sequential selection of two species of
bph
deletion derivatives occurs at high frequency. This mirrors observations made with TOL plasmids (encoding toluene and xylene catabolism) grown under similar conditions. Subcloning of dioxygenase genes involved in biphenyl catabolism confirmed the localization of the
bph
genes on the wild-type plasmid and the RP4 cointegrate plasmid.
Biotransformation of the Herbicide Atrazine by the White Rot Fungus
Phanerochaete chrysosporium
Authors: Mougin Christian , Laugero Chantal , Asther Michele , Dubroca Jacqueline , Frasse Pierre , Asther Marcel
Published: January 2020

Biotransformation of atrazine by the white rot fungus
Phanerochaete chrysosporium
was demonstrated by a 48% decrease of the initial herbicide concentration in the growth medium within the first 4 days of incubation, which corresponded to the mycelium-growing phase. Results clearly established the mineralization of the ethyl group of the herbicide. Analysis of the growth medium showed the formation of hydroxylated and/or N-dealkylated metabolites of atrazine during fungal degradation.
Stability and Activities of Antibiotics Produced during Infection of the Insect
Galleria mellonella
by Two Isolates of
Xenorhabdus nematophilus
Authors: Maxwell Philip W. , Chen Genhui , Webster John M. , Dunphy Gary B.
Published: January 2020

Xenorhabdus nematophilus
subsp.
dutki
, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect
Galleria mellonella
may contribute to assessing the possible impact of antibiotics on soil bacteria. Two isolates of
X. nematophilus
subsp.
dutki
(isolates GI and SFU) produced xenocoumacins 1 and 2 in cadavers of
G. mellonella
larvae in a 1:1 ratio. Total xenocoumacin 1 and 2 production was 800 ng/200 mg (wet weight) of insect tissue for the GI isolate. Antibiotic activity of water extracts from insects that had been infected with
X. nematophilus
was stable at 60°C for 1 h and after repeated freeze-thaw cycles. The antibiotic titer of extracts held at 27°C declined by day 10. The spectrum of bacterial species killed by antibiotics produced in insect cadavers varied with the isolate of
X. nematophilus
. Levels of antibiotic activity were greater in vivo than in tryptic soy broth, which may represent a nutrient effect. The bacterial isolate, culture condition, and presence of nematodes influenced the total antibiotic production in vivo. However, the levels of activity were not correlated with bacterial levels in the different growth environments. Insect cadavers with antibiotic activity transiently lowered the numbers of the bacteria in the soil, the extent of decline varying with the strain of
X. nematophilus
and the time of sampling.
Sensitivity of Iron-Oxidizing Bacteria,
Thiobacillus ferrooxidans
and
Leptospirillum ferrooxidans
, to Bisulfite Ion
Authors: Sugio Tsuyoshi , Uemura Satomi , Makino Ikuko , Iwahori Kenji , Tano Tatsuo , Blake Robert C.
Published: January 2020

When grown on iron-salt medium supplemented with the bisulfite ion,
Leptospirillum ferrooxidans
was much more sensitive to the ion than was
Thiobacillus ferrooxidans
. The causes of the sensitivity of
L. ferrooxidans
to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of
L. ferrooxidans
and
T. ferrooxidans
at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of
T. ferrooxidans
. However, the treatment of
L. ferrooxidans
with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity.
T. ferrooxidans
had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in
L. ferrooxidans
, suggesting that it is much more difficult for
L. ferrooxidans
to oxidize the bisulfite ion to the less harmful sulfate than it is for
T. ferrooxidans
. These results suggest that the sensitivity of
L. ferrooxidans
to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.
A Role for Exopolysaccharides in the Protection of Microorganisms from Desiccation
Authors: Ophir Tslil , Gutnick David L.
Published: January 2020

Mucoid strains of
Escherichia coli, Acinetobacter calcoaceticus
, and
Erwinia stewartii
were significantly more resistant to desiccation than corresponding isogenic nonmucoid mutants (survival rates of up to 35% in mucoid strains and between 0.7 and 5% in nonmucoid variants), even in colonies containing both cell types. Desiccation was found to bring about an induction of β-galactosidase in Lon
-
strains of
E. coli
K-12 carrying transcriptional
lac
fusions in the capsule biosynthetic (
cps
) regulon. This induction was dependent on the transcriptional activators RcsA and RcsB. Induction was lower in cells carrying mutations in the membrane sensor protein RcsC.
Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms
Authors: Kopczynski E D , Bateson M M , Ward D M
Published: January 2020

When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.
Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washington
Authors: Samadpour M , Ongerth J E , Liston J , Tran N , Nguyen D , Whittam T S , Wilson R A , Tarr P I
Published: January 2020

Fresh meat, poultry, and seafood purchased from Seattle area grocery stores were investigated for the presence of Shiga-like toxin-producing Escherichia coli by using DNA probes for Shiga-like toxin (SLT) genes I and II. Of the 294 food samples tested, 17% had colonies with sequence homology to SLT I and/or SLT II genes.
Field Evaluation of Two Colorimetric Coliphage Detection Methods
Authors: Ijzerman M. Marian , Falkinham Joseph O. , Reneau Raymond B. , Hagedorn Charles
Published: January 2020

Two new methods for coliphage detection, a colorimetric agar-based (CAB) method and a liquid colorimetric presence-absence (LCPA) method, were compared to the coliphage method proposed by the American Public Health Association (APHA;
Standard Methods for the Examination of Water and Wastewater
, 18th ed., American Public Health Association, Washington, D.C., 1992). Both new methods are based on the induction of β-galactosidase in
Escherichia coli
and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate which produces a colored reaction product. Ninety split water samples from four different sources were tested. A total of 52 samples were positive by the CAB method, 52 were positive by the LCPA method, and 53 were positive by the APHA method. Results indicated that (i) the CAB and LCPA methods were as sensitive in coliphage detection as the APHA method, (ii) both the CAB and LCPA methods were easier to read and interpret than the APHA method, and (iii) the CAB method detected more coliphages in a positive sample than the APHA method in two of the four types of water sources. Importantly, the rapid and simple LCPA method was as reliable and sensitive as either of the two agar-based methods in coliphage detection.
Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges
Authors: Top Eva , De Smet Ingrid , Verstraete Willy , Dijkmans Roger , Mergeay Max
Published: January 2020

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with
Escherichia coli
(a member of the γ subgroup of the
Proteobacteria
) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes
czc
: Co
r
, Zn
r
, and Cd
r
),
Alcaligenes eutrophus
(a member of the β subgroup of the
Proteobacteria
) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (
E. coli
CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.
Extracellular enzyme activity in anaerobic bacterial cultures: evidence of pullulanase activity among mesophilic marine bacteria
Authors: Arnosti C , Repeta D J
Published: January 2020

The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of > or = 10,000, approximately 5,000, and < or = 1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at alpha(1,6) linkages by pullulanase enzymes, most likely type II pullulanase. Although isolated pullulanase enzymes have been shown to hydrolyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino, and R. M. Kelly, Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Klingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-152, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked through one alpha(1,6) linkage. Either the final hydrolysis step was closely linked to substrate uptake, or specialized porins similar to maltoporin might permit direct transport of large oligosaccharides into the bacterial cell. This is the first report of pullulanase activity among mesophilic marine bacteria. The combination of GPC and NMR could easily be used to assess other types of extracellular enzyme activity in bacterial cultures.
Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism
Authors: Dabrock B , Kesseler M , Averhoff B , Gottschalk G
Published: January 2020

Rhodococcus erythropolis BD2, which is able to utilize isopropylbenzene as a sole carbon and energy source, was shown to contain a conjugative linear plasmid, pBD2. The estimated size of pBD2 is 208 to 212 kb. Linear plasmid-deficient strains had lost both the isopropylbenzene degradation and trichloroethene degradation characteristics, as well as the arsenite resistance and mercury resistance phenotypes. Reintroduction of pBD2 restored all four characteristics. Conjugational transfer of pBD2 to a plasmidless mutant of strain BD2 and other R. erythropolis strains occurred at frequencies between 3.5 x 10(-5) and 2.6 x 10(-3) transconjugants per recipient. R. erythropolis BD2 degrades isopropylbenzene via 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate. Both isopropylbenzene-oxidizing and meta-cleavage activities were shown to correspond with the presence of pBD2. Southern hybridizations with DNA encoding the toluene dioxygenase structural genes (todC1C2BA) from Pseudomonas putida F1 revealed homology to linear plasmid DNA. These results indicate that the isopropylbenzene degradation pathway encoded by linear plasmid pBD2 is initiated by an isopropylbenzene dioxygenase analogous to toluene dioxygenase.
Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii
Authors: Moyer C L , Dobbs F C , Karl D M
Published: January 2020

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity.
Cloning, sequencing, and expression in Escherichia coli of the D-hydantoinase gene from Pseudomonas putida and distribution of homologous genes in other microorganisms
Authors: LaPointe G , Viau S , LeBlanc D , Robert N , Morin A
Published: January 2020

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The gene encoding this D-hydantoinase enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of 1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regions of identity between the predicted amino acid sequence and that of other known amidohydrolases (two other D-hydantoinases, allantionase and dihydroorotase) were found. The D-hydantoinase gene was used as a probe to screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe was specific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria, or plants known to express D-hydantoinase activity.
Distribution of tetracycline resistance determinants among gram-negative bacteria isolated from polluted and unpolluted marine sediments
Authors: Andersen S R , Sandaa R A
Published: January 2020

Tetracycline-resistant gram-negative bacteria were isolated from four different marine sediments in Scandinavia and analyzed with DNA probes for the determinant classes A to E. Colony hybridizations of 429 isolates revealed that class E is the dominating resistance determinant in these marine sediments. Comparison of fecally polluted and unpolluted sediments showed few determinant classes in unpolluted sediment and a complex composition of several determinant classes in polluted sediment. Total DNA extraction and analysis with DNA probes for determinant classes A to E resulted in no hybridization signal, because of the low number of gram-negative tetracycline-resistant bacteria. Identification of class E isolates revealed that this determinant is present not only in Aeromonas hydrophila, Escherichia coli, and Vibrio salmonicida but also in additional strains.
Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains
Authors: Rosselló-Mora R A , Lalucat J , García-Valdés E
Published: January 2020

Of a 49-strain collection of Pseudomonas stutzeri species, 11 isolates were able to degrade naphthalene and 1 isolate was able to use m- and p-toluate as sole carbon and energy sources. Of these 12 strains, 10 shared a highly homologous set of naphthalene catabolic genes, even though they belong to four different genomovars. These genes differed from those present in plasmid NAH7. In only one of these degraders could a plasmid-encoded pathway be demonstrated, and a chromosome-encoded pathway is proposed for the remaining strains. meta cleavage of catechol was only observed in those strains able to metabolize alkyl derivatives of catechol.
Airborne environmental endotoxin: a cross-validation of sampling and analysis techniques
Authors: Walters M , Milton D , Larsson L , Ford T
Published: January 2020

A standard method for measurement of airborne environmental endotoxin was developed and field tested in a fiberglass insulation-manufacturing facility. This method involved sampling with a capillary-pore membrane filter, extraction in buffer using a sonication bath, and analysis by the kinetic-Limulus assay with resistant-parallel-line estimation (KLARE). Cross-validation of the extraction and assay method was performed by comparison with methanolysis of samples followed by 3-hydroxy fatty acid (3-OHFA) analysis by gas chromatography-mass spectrometry. Direct methanolysis of filter samples and methanolysis of buffer extracts of the filters yielded similar 3-OHFA content (P = 0.72); the average difference was 2.1%. Analysis of buffer extracts for endotoxin content by the KLARE method and by gas chromatography-mass spectrometry for 3-OHFA content produced similar results (P = 0.23); the average difference was 0.88%. The source of endotoxin was gram-negative bacteria growing in recycled washwater used to clean the insulation-manufacturing equipment. The endotoxin and bacteria become airborne during spray cleaning operations. The types of 3-OHFAs in bacteria cultured from the washwater, present in the washwater and in the air, were similar. Virtually all of the bacteria cultured from air and water were gram negative composed mostly of two species, Deleya aesta and Acinetobacter johnsonii. Airborne countable bacteria correlated well with endotoxin (r2 = 0.64). Replicate sampling showed that results with the standard sampling, extraction, and Limulus assay by the KLARE method were highly reproducible (95% confidence interval for endotoxin measurement +/- 0.28 log10). These results demonstrate the accuracy, precision, and sensitivity of the standard procedure proposed for airborne environmental endotoxin.
Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria
Authors: Leite R P , Minsavage G V , Bonas U , Stall R E
Published: January 2020

Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv. vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria. DNA sequences related to the hrp genes were successfully amplified from X. fragariae and from 28 pathovars of X. campestris. No DNA amplification occurred with genomic DNA from phytopathogenic strains of X. campestris pv. secalis, X. campestris pv. translucens, and X. albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella. The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis. DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads. However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains. The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads. We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads.
Restriction fragment length polymorphism evidence for genetic homology within a pathovar of Pseudomonas syringae
Authors: Scholz B K , Jakobek J L , Lindgren P B
Published: January 2020

Pseudomonas syringae pv. phaseolicola NPS3121 hrp sequences were used as hybridization probes in a restriction fragment length polymorphism (RFLP) analysis of 24 P. syringae pv. tabaci strains as a means to evaluate the genetic and taxonomic relationship of pathovars of P. syringae. Southern blot analyses of genomic restriction digests, with hrpA-S sequences as hybridization probes, and restriction analyses of PCR-amplified DNA of regions within hrpD were conducted. The resulting RFLP patterns were uniform for 23 of the 24 isolates tested, with strain BR2R having a unique pattern. BR2R is a pathogen of bean which was classified as pathovar tabaci because of its ability to produce tabtoxin, but unlike the other 23 tabaci strains in this study, it does not incite disease symptoms on tobacco. When a DNA fragment containing hrpM sequences was used as a hybridization probe, the tabaci isolates could be divided into three groups on the basis of the RFLP patterns : BR2R, Pt11528R and Pt113R, and the remaining strains. For all of the above analyses, BR2R shared identical RFLP patterns with P. syringae pv. phaseolicola NPS3121, also a bean pathogen which does not cause disease on tobacco. However, BR2R AND NPS3121 could be differentiated from each other on the basis of the RFLP patterns from restriction analysis of PCR-amplified DNA of argF, while the remaining tabaci strains had a third pattern. These studies indicate that hrp genes and argF are conserved in strains of P. syringae pathogenic to tobacco, suggesting that P. syringae strains pathogenic to specific hosts may have a high level of genetic similarity. We believe that these analyses have shown that distinct identifiable genetic differences may be correlated with host range and suggest that such information may be useful for assigning pathovar designations.
Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils
Authors: Ka J O , Holben W E , Tiedje J M
Published: January 2020

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of competition in soil among 2,4-dichlorophenoxyacetic acid-degrading bacteria
Authors: Ka J O , Holben W E , Tiedje J M
Published: January 2020

Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions. The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712. Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S. paucimobilis 1443, which belongs to a different class of 2,4-D degraders. P. cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations. The S. paucimobilis population was the secondary dominant population, and strain 745 and P. pickettii were not detected. Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains. Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome. Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P. cepacia DBO1 resulted in the slower growth characteristic of its original host, P. pickettii, rather than the rapid growth observed when this strain harbors pJP4.
Variation of Clonal, Mesquite-Associated Rhizobial and Bradyrhizobial Populations from Surface and Deep Soils by Symbiotic Gene Region Restriction Fragment Length Polymorphism and Plasmid Profile Analysis
Authors: Thomas P. M. , Golly K. F. , Zyskind J. W. , Virginia R. A.
Published: January 2020

Genetic characteristics of 14
Rhizobium
and 9
Bradyrhizobium
mesquite (
Prosopis glandulosa
)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of
Pst
I-digested genomic DNA were sequentially hybridized with the
nodDABC
region of
Rhizobium meliloti
, the
Klebsiella pneumoniae nifHDK
region encoding nitrogenase structural genes, and the chromosome-localized
ndvB
region of
R. meliloti.
Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (
R. meliloti, Rhizobium leguminosarum
bv. phaseoli, and
Parasponia bradyrhizobia
). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The
ndvB
hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of
ndvB
-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with
nifHDK
and
nodDABC
probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity.
Comparison of the microbiological quality of water coolers and that of municipal water systems
Authors: Lévesque B , Simard P , Gauvin D , Gingras S , Dewailly E , Letarte R
Published: January 2020

The microbiological quality of tap water and that of water from 50 water coolers located in residences and workplaces were comparatively studied. In addition, difference factors that might influence the bacteriological contamination of water dispensers were examined. Aeorbic and facultative anaerobic heterotrophic bacteria, total coliforms, and two indicators for fecal contamination (fecal coliforms and fecal streptococci) as well as three types of pathogenic bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, and Aeromonas spp.) were enumerated. It was found that 36 and 28% of the water dispenser samples from the residences and the workplaces, respectively, were contaminated by a least one coliform or indicator bacterium and/or at least one pathogenic bacterium. The respective proportions of tap water samples contaminated in a similar fashion were 18 and 22%, much less than those observed for water coolers (Chi2(1) = 3.71, P = 0.05). We were unable to discern the dominant factors responsible for the contamination of water coolers, but cleaning the water dispenser every 2 months seemed to limit the extent of contamination.
Generation and Characterization of Environmentally Sensitive Variants of the β-Galactosidase from
Lactobacillus delbrueckii
subsp.
bulgaricus
Authors: Yoast Sienna , Adams Robin M. , Mainzer Stanley E. , Moon Keith , Palombella Anthony L. , Schmidt Brian F.
Published: January 2020

A method is described for generating and screening variants of the β-galactosidase from
Lactobacillus delbrueckii
subsp.
bulgaricus
sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the β-galactosidase gene carried on an
Escherichia coli
expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on β-galactosidase indicator plates. The mutations responsible for three variant β-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive.