bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidation of Methyl Halides by the Facultative Methylotroph Strain IMB-1
Authors: Schaefer Jeffra K. , Oremland Ronald S. 
Published: December 2019

ABSTRACT

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 μM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [
14
C]formaldehyde to
14
CO
2
but had only a small capacity for oxidation of [
14
C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [
14
C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent

K
s

values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO
2
. The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.
Reactor-Scale Cultivation of the Hyperthermophilic Methanarchaeon
Methanococcus jannaschii
to High Cell Densities
Authors: Mukhopadhyay Biswarup , Johnson Eric F. , Wolfe Ralph S. 
Published: December 2019

ABSTRACT

For the hyperthermophilic and barophilic methanarchaeon
Methanococcus jannaschii
, we have developed a medium and protocols for reactor-scale cultivation that improved the final cell yield per liter from ∼0.5 to ∼7.5 g of packed wet cells (∼1.8 g dry cell mass) under autotrophic growth conditions and to ∼8.5 g of packed wet cells (∼2 g dry cell mass) with yeast extract (2 g liter
−1
) and tryptone (2 g liter
−1
) as medium supplements. For growth in a sealed bottle it was necessary to add Se to the medium, and a level of 2 μM for added Se gave the highest final cell yield. In a reactor
M. jannaschii
grew without added Se in the medium; it is plausible that the cells received Se as a contaminant from the reactor vessel and the H
2
S supply. But, for the optimal performance of a reactor culture, an addition of Se to a final concentration of 50 to 100 μM was needed. Also, cell growth in a reactor culture was inhibited at much higher Se concentrations. These observations and the data from previous work with methanogen cell extracts (B. C. McBride and R. S. Wolfe, Biochemistry 10:4312–4317, 1971) suggested that from a continuously sparged reactor culture Se was lost in the exhaust gas as volatile selenides, and this loss raised the apparent required level of and tolerance for Se. In spite of having a proteinaceous cell wall,
M. jannaschii
withstood an impeller tip speed of 235.5 cms
−1
, which was optimal for achieving high cell density and also was the higher limit for the tolerated shear rate. The organism secreted one or more acidic compounds, which lowered pH in cultures without pH control; this secretion continued even after cessation of growth.
Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments
Authors: Costello Andria M. , Lidstrom Mary E. 
Published: December 2019

ABSTRACT

The 16S rRNA and
pmoA
genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes
Msp
I,
Hae
III, and
Hha
I. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera
Methylomonas
,
Methylosinus
, and
Methylocystis
were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs,
pmoA
. Phylogenetic analyses of both the 16S rRNA and
pmoA
gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.
Natural Communities of
Achromatium oxaliferum
Comprise Genetically, Morphologically, and Ecologically Distinct Subpopulations
Authors: Gray N. D. , Howarth R. , Rowan A. , Pickup R. W. , Jones J. Gwyn , Head I. M. 
Published: December 2019

ABSTRACT

The diversity and ecology of natural communities of the uncultivated bacterium
Achromatium oxaliferum
were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as
A. oxaliferum
present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the
A. oxaliferum
communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified
A. oxaliferum
cells corresponded to genetically distinct
Achromatium
subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the
Achromatium
sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between
Achromatium
communities from different freshwater sediments, the distribution of different subpopulations of
Achromatium
in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that
Achromatium
populations have undergone adaptive radiation and that the divergent
Achromatium
species occupy different niches in the sediments which they inhabit.
Analyses of Spatial Distributions of Sulfate-Reducing Bacteria and Their Activity in Aerobic Wastewater Biofilms
Authors: Okabe Satoshi , Itoh Tsukasa , Satoh Hisashi , Watanabe Yoshimasa 
Published: December 2019

ABSTRACT

The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating disk reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O
2
, H
2
S, NO
2
−
, NO
3
−
, NH
4
+
, and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 10
9
to 10
10
cells per cm
3
of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained
Desulfobulbus
spp. were found to be numerically important members of SRB populations (approximately 10
8
to 10
9
cells per cm
3
). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 μm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S
0
) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 μm), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate.
Reductive Dehalogenation and Conversion of 2-Chlorophenol to 3-Chlorobenzoate in a Methanogenic Sediment Community: Implications for Predicting the Environmental Fate of Chlorinated Pollutants
Authors: Becker Jennifer G. , Stahl David A. , Rittmann Bruce E. 
Published: December 2019

ABSTRACT

Biotransformation of 2-chlorophenol by a methanogenic sediment community resulted in the transient accumulation of phenol and benzoate. 3-Chlorobenzoate was a more persistent product of 2-chlorophenol metabolism. The anaerobic biotransformation of phenol to benzoate presumably occurred via
para
-carboxylation and dehydroxylation reactions, which may also explain the observed conversion of 2-chlorophenol to 3-chlorobenzoate.
Epitope Identification for a Panel of Anti-
Sinorhizobium meliloti
Monoclonal Antibodies and Application to the Analysis of K Antigens and Lipopolysaccharides from Bacteroids
Authors: Reuhs Bradley L. , Stephens Samuel B. , Geller Daniel P. , Kim John S. , Glenn Joshua , Przytycki Jessica , Ojanen-Reuhs Tuula 
Published: December 2019

ABSTRACT

In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of
Sinorhizobium meliloti
were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506–509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654–661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-
S. meliloti
MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from
S. meliloti
and
Sinorhizobium fredii
. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from
S. meliloti
bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of
S. meliloti
NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the
S. meliloti
NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.
Ubiquity and Diversity of Dissimilatory (Per)chlorate-Reducing Bacteria
Authors: Coates John D. , Michaelidou Urania , Bruce Royce A. , O’Connor Susan M. , Crespi Jill N. , Achenbach Laurie A. 
Published: December 2019

ABSTRACT

Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 × 10
3
to 2.4 × 10
6
cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type
c
cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 μmol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the
Proteobacteria
. Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera
Pseudomonas
and
Azospirllum
. However, many were not closely related to any previously described organism and represented new genera within the
Proteobacteria
. The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.
Characterization of Unexpected Growth of
Escherichia coli
O157:H7 by Modeling
Authors: Cornu Marie , Delignette-Muller Marie Laure , Flandrois Jean-Pierre 
Published: December 2019

ABSTRACT

Modeling of batch kinetics in minimal synthetic medium was used to characterize
Escherichia coli
O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for
E. coli
K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other
E. coli
strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures,
E. coli
K-12 had a positive effect on
E. coli
O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for
E. coli
K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of
E. coli
K-12 on
E. coli
O157:H7 appeared to be similar to the effect of methionine.
Influence of Different Functional Elements of Plasmid pGT232 on Maintenance of Recombinant Plasmids in
Lactobacillus reuteri
Populations In Vitro and In Vivo
Authors: Heng Nicholas C. K. , Bateup Judith M. , Loach Diane M. , Wu Xiyang , Jenkinson Howard F. , Morrison Mark , Tannock Gerald W. 
Published: December 2019

ABSTRACT

Plasmid pGT232 (5.1 kb), an indigenous plasmid of
Lactobacillus reuteri
100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein,
repA
. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an
L. reuteri
population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the
L. reuteri
population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.
Fate of pGFP-Bearing
Escherichia coli
O157:H7 in Ground Beef at 2 and 10°C and Effects of Lactate, Diacetate, and Citrate
Authors: Ajjarapu Srilatha , Shelef Leora A. 
Published: December 2019

ABSTRACT

Although beef has been implicated in the largest outbreaks of
Escherichia coli
O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing
E. coli
O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10°C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2°C, the initial numbers of
E. coli
O157:H7 (3 and 5 log
10
CFU/g) decreased by ∼1 log
10
CFU/g when spoilage was manifest (>7 log
10
CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10°C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.
Precise Detection and Tracing of
Trichoderma hamatum
382 in Compost-Amended Potting Mixes by Using Molecular Markers
Authors: Abbasi Pervaiz A. , Miller Sally A. , Meulia Tea , Hoitink Harry A. J. , Kim Jin-Man 
Published: December 2019

ABSTRACT

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of
Trichoderma hamatum
382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of
T. hamatum
382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16
0.35
), 0.6 (OPH-19
0.6
), and 0.65 (OPH-20
0.65
) kb were diagnostic for
T. hamatum
382, clearly distinguishing it from 53 isolates of four other
Trichoderma
spp. tested. Some isolates of
T. hamatum
shared these low-molecular-weight fragments with
T. hamatum
382. However, RAPD analysis of isolates of
T. hamatum
with all three random primers used in consecutive PCR tests distinguished
T. hamatum
382 from other isolates of
T. hamatum
. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of
T. hamatum
382. A combination of dilution plating on a semiselective medium for
Trichoderma
spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of
T. hamatum
382 propagules in nine different soil, compost, and potting mix samples. All 23
Trichoderma
isolates recovered on semiselective medium from commercial potting mixes fortified with
T. hamatum
382 were identified as
T. hamatum
382, whereas 274
Trichoderma
isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of
T. hamatum
382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of
T. hamatum
382 directly from fortified compost-amended potting mixes.
Cloning, Sequencing, and Characterization of Genomic Subtracted Sequences from
Listeria monocytogenes
Authors: Wu Fone-Mao , Muriana Peter M. 
Published: December 2019

ABSTRACT

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for
Listeria monocytogenes
but did not hybridize with members of other bacterial genera. Sequence analysis identified several
L. monocytogenes
sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.