bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Methanogens and Other Bacteria in Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments
Authors: Lomans Bart P. , Op den Camp Huub J. M. , Pol Arjan , van der Drift Chris , Vogels Godfried D.
Published: December 2019

The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 μmol of DMS was stoichiometrically converted into 112 μmol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.
Construction and Characterization of Two Recombinant Bacteria That Grow on
ortho
- and
para
-Substituted Chlorobiphenyls
Authors: Hrywna Yarek , Tsoi Tamara V. , Maltseva Olga V. , Quensen John F. , Tiedje James M.
Published: December 2019

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing
Comamonas testosteroni
VP44 resulted in recombinant pathways allowing growth on
ortho
- and
para
-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the
Pseudomonas aeruginosa
142
ohb
genes coding for the terminal oxygenase (ISP
OHB
) of the
ortho
-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the
Arthrobacter globiformis
KZT1
fcb
genes, which catalyze the hydrolytic
para
-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.
Genetic Diversity of African and Worldwide Strains of
Ralstonia solanacearum
as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the
hrp
Gene Region
Authors: Poussier Stephane , Vandewalle Peggy , Luisetti Jacques
Published: December 2019

The genetic diversity among a worldwide collection of 120 strains of
Ralstonia solanacearum
was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the
hrp
gene region. Five amplified fragments appeared to be specific to
R. solanacearum
. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the
hrp
gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.
Differentiation of
Methanosaeta concilii
and
Methanosarcina barkeri
in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy
Authors: Rocheleau Sylvie , Greer Charles W. , Lawrence John R. , Cantin Christiane , Laramée Louise , Guiot Serge R.
Published: December 2019

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate
Methanosaeta concilii, Methanosarcina barkeri
, and mesophilic methanogens. All
M. concilii
oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific
M. concilii
oligonucleotide probe.
Methanosarcina barkeri
oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different
Methanosarcina
species. The MB4 probe specifically detected
Methanosarcina barkeri
, and the MB3 probe detected the presence of all mesophilic
Methanosarcina
species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of
M. concilii
and
Methanosarcina barkeri
, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of
M. concilii
. In contrast, a layered structure developed in methanol-enriched granules, where
M. barkeri
growth was induced in an outer layer. This outer layer was followed by a layer composed of
M. concilii
, with an inner core of
M. concilii
and other bacteria.
Detection of Bacteriocins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Authors: Rose Natisha L. , Sporns Peter , McMullen Lynn M.
Published: December 2019

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of bacteriocins was investigated. A 30-s water wash of the sample on the MALDI-TOF MS probe was effective in removing contaminants of the analyte. This method was used for rapid detection of nisin, pediocin, brochocin A and B, and enterocin A and B from culture supernatants and for detection of enterocin B throughout its purification.
A PCR Detection Method for Rapid Identification of
Paenibacillus larvae
Authors: Govan V. A. , Allsopp M. H. , Davison S.
Published: December 2019

American foulbrood is a disease of larval honeybees (
Apis mellifera
) caused by the bacterium
Paenibacillus larvae
. Over the years attempts have been made to develop a selective medium for the detection of
P. larvae
spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of
P. larvae
and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of
P. larvae
. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of
P. larvae
. The PCR primers were designed on the basis of the 16S rRNA gene of
P. larvae
and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from
P. larvae
by sequencing in both directions. Detection was specific for
P. larvae
, and the primers did not hybridize with DNA from closely related bacterial species.
Microbial Biomass and Activity in Lead-Contaminated Soil
Authors: Konopka A. , Zakharova T. , Bischoff M. , Oliver L. , Nakatsu C. , Turco R. F.
Published: December 2019

Microbial community diversity, potential microbial activity, and metal resistance were determined in three soils whose lead contents ranged from 0.00039 to 48 mmol of Pb kg of soil
−1
. Biomass levels were directly related to lead content. A molecular analysis of 16S rRNAs suggested that each soil contained a complex, diverse microbial community. A statistical analysis of the phospholipid fatty acids indicated that the community in the soil having the highest lead content was not related to the communities in the other soils. All of the soils contained active microbial populations that mineralized [
14
C]glucose. In all samples, 10 to 15% of the total culturable bacteria were Pb resistant and had MIC of Pb for growth of 100 to 150 μM.
Divergence of West African and North American Communities of
Aspergillus
Section
Flavi
Authors: Cotty Peter J. , Cardwell Kitty F.
Published: December 2019

West African
Aspergillus flavus
S isolates differed from North American isolates. Both produced aflatoxin B
1
. However, 40 and 100% of West African isolates also produced aflatoxin G
1
in NH
4
medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G
1
. This geographical and physiological divergence may influence aflatoxin management.
Transcriptional Control of
ADH
Genes in the Xylose-Fermenting Yeast
Pichia stipitis
Authors: Cho Jae-Yong , Jeffries Thomas W.
Published: December 2019

We studied the expression of the genes encoding group I alcohol dehydrogenases (
PsADH1
and
PsADH2
) in the xylose-fermenting yeast
Pichia stipitis
CBS 6054. The cells expressed
PsADH1
approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of
PsADH2
were not detectable under either aeration condition. We used a
PsADH1
::
lacZ
fusion to monitor
PsADH1
expression and found that expression increased as oxygen decreased. The level of
PsADH1
transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of
PsADH1
,
PsCYC1
expression was repressed. These results indicate that oxygen availability regulates
PsADH1
expression and that regulation may be mediated by heme. The regulation of
PsADH2
expression was also examined in other genetic backgrounds. Disruption of
PsADH1
dramatically increased
PsADH2
expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of
PsADH2
is subject to feedback regulation under these conditions.
Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria
Authors: Daane L. L. , Häggblom M. M.
Published: December 2019

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found thatRalstonia eutropha(pJP4) can be recovered fromEisenia fetidacocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence ofR. eutropha(pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containingR. eutropha(pJP4) at either low or high densities (102or 105CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 104CFUR. eutropha(pJP4) g−1of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or withoutR. eutropha(pJP4). Results showed that cocoons containingR. eutropha(pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP byR. eutropha(pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.
Flow Cytometric Analysis of 5-Cyano-2,3-Ditolyl Tetrazolium Chloride Activity of Marine Bacterioplankton in Dilution Cultures
Authors: Sieracki Michael E. , Cucci Terry L. , Nicinski Justyna
Published: December 2019

The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not “activate” a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.
Environmental Factors Modulating Antibiotic and Siderophore Biosynthesis by
Pseudomonas fluorescens
Biocontrol Strains
Authors: Duffy Brion K. , Défago Geneviève
Published: December 2019

Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of
Pseudomonas fluorescens
is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn
2+
, NH
4
Mo
2+
, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn
2+
, Co
2+
, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn
2+
and NH
4
Mo
2+
. Pyochelin production was increased by Co
2+
, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH
4
Mo
2+
, glycerol, and glucose. The mixture of Zn
2+
and NH
4
Mo
2+
with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41
P. fluorescens
biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn
2+
and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn
2+
and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.
Enhancement of Expression and Apparent Secretion of
Erwinia chrysanthemi
Endoglucanase (Encoded by
celZ
) in
Escherichia coli
B
Authors: Zhou Shengde , Yomano Lorraine P. , Saleh Alif Z. , Davis Francis C. , Aldrich Henry C. , Ingram Lonnie O.
Published: December 2019

Escherichia coli
B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of
E. coli
B can produce high levels of
Erwinia chrysanthemi
endoglucanase (encoded by
celZ
) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve
celZ
expression in
E. coli
B. Initially, with a low-copy-number vector, two
E. coli
glycolytic gene promoters (
gap
and
eno
) were tested and found to be less effective than the original
celZ
promoter. By screening 18,000 random fragments of
Zymomonas mobilis
DNA, a surrogate promoter was identified which increased
celZ
expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five
Sau
3A1 fragments, one of which was previously sequenced in
Z. mobilis
. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the
out
genes from
E. chrysanthemi
EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.
Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from
Aspergillus niger
Authors: de Vries Ronald P. , van den Broeck Hetty C. , Dekkers Ester , Manzanares Paloma , de Graaff Leo H. , Visser Jaap
Published: December 2019

A gene encoding a third α-galactosidase (AglB) from
Aspergillus niger
has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes
A. niger
AglA.
A. niger
AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of
aglA
,
aglB
,
aglC
, and
lacA
, the latter of which encodes an
A. niger
β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of
aglA
is only detected on galactose and galactose-containing oligomers and polymers. The
aglB
gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of
aglC
was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of
lacA
was detected on arabinose, xylose, xylan, and pectin. Similar to
aglB
, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.
Monooxygenase-Mediated 1,2-Dichloroethane Degradation by
Pseudomonas
sp. Strain DCA1
Authors: Hage Jacobus C. , Hartmans Sybe
Published: December 2019

A bacterial strain, designated
Pseudomonas
sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a

K
m

value below the detection limit of 0.5 μM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.
Maintenance Energy Demand and Starvation Recovery Dynamics ofNitrosomonas europaeaandNitrobacter winogradskyiCultivated in a Retentostat with Complete Biomass Retention
Authors: Tappe W. , Laverman A. , Bohland M. , Braster M. , Rittershaus S. , Groeneweg J. , van Verseveld H. W.
Published: December 2019

Nitrosomonas europaeaandNitrobacter winogradskyi(strain “Engel”) were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25°C and pH 8. Fitting the retentostat biomass and oxygen consumption data ofN. europaeaandN. winogradskyito the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 μmol of O2mg−1of biomass-C · h−1for bothN. europaeaandN. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster forN. winogradskyithan forN. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable.N. europaeamore readily recovers from starvation thanN. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 μg · ml−1) inhibition experiments withN. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514–1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.
Microbial System for Polysaccharide Depolymerization: Enzymatic Route for Xanthan Depolymerization by
Bacillus
sp. Strain GL1
Authors: Nankai Hirokazu , Hashimoto Wataru , Miki Hikaru , Kawai Shigeyuki , Murata Kousaku
Published: December 2019

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in
Bacillus
sp. strain GL1, which was closely related to
Brevibacillus thermoruber
, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765–3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular β-
d
-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by β-
d
-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by α-
d
-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel β-
d
-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45°C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.
Cellobiose Transport by Mixed Ruminal Bacteria from a Cow
Authors: Kajikawa Hiroshi , Masaki Shigehiko
Published: December 2019

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the

K
m

and 10 nmol/min/mg of protein for the
V
max
. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher
V
max
values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.
Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria
Authors: Reay David S. , Nedwell David B. , Priddle Julian , Ellis-Evans J. Cynan
Published: December 2019

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures.
Niche-Partitioning of
Prochlorococcus
Populations in a Stratified Water Column in the Eastern North Atlantic Ocean
Authors: West Nyree J. , Scanlan David J.
Published: December 2019

The in situ community structure of
Prochlorococcus
populations in the eastern North Atlantic Ocean was examined by analysis of
Prochlorococcus
16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL)
Prochlorococcus
genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of
Prochlorococcus
genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted
Prochlorococcus
strains. DGGE analysis and the sequencing of
Prochlorococcus
16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of
Prochlorococcus
genotypes in a stratified water column provide a genetic basis for the dim and bright
Prochlorococcus
populations observed in flow cytometric signatures in several oceanic provinces.
Vibrios Associated with
Litopenaeus vannamei
Larvae, Postlarvae, Broodstock, and Hatchery Probionts
Authors: Vandenberghe Johan , Verdonck Linda , Robles-Arozarena Rocio , Rivera Gabriel , Bolland Annick , Balladares Marcos , Gomez-Gil Bruno , Calderon Jorge , Sorgeloos Patrick , Swings Jean
Published: December 2019

Several bacteriological surveys were performed from 1994 to 1996 at different
Litopenaeus vannamei
hatcheries (in Ecuador) and shrimp farms (in Mexico). Samples were taken from routine productions of healthy and diseased
L. vannamei
larvae, postlarvae, and their culture environment and from healthy and diseased juveniles and broodstock. In Ecuador, the dominant bacterial flora associated with shrimp larvae showing symptoms of zoea 2 syndrome, mysis mold syndrome, and bolitas syndrome has been determined. Strains were characterized by Biolog metabolic fingerprinting and identified by comparison to a database of 850
Vibrio
type and reference strains. A selection of strains was further genotypically fine typed by AFLP.
Vibrio alginolyticus
is predominantly present in all larval stages and is associated with healthy nauplius and zoea stages. AFLP genetic fingerprinting shows high genetic heterogeneity among
V. alginolyticus
strains, and the results suggest that putative probiotic and pathogenic strains each have specific genotypes.
V. alginolyticus
was found to be associated with larvae with the zoea 2 syndrome and the mysis mold syndrome, while different
Vibrio
species (
V. alginolyticus
and
V. harveyi
) are associated with the bolitas syndrome.
V. harveyi
is associated with diseased postlarvae, juveniles, and broodstock. The identities of the strains identified as
V. harveyi
by the Biolog system could not be unambiguously confirmed by AFLP genomic fingerprinting.
Vibrio
strain STD3-988 and one unidentified strain (STD3-959) are suspected pathogens of only juvenile and adult stages.
V. parahaemolyticus
,
Photobacterium damselae
, and
V. mimicus
are associated with juvenile and adult stages.
Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis
Authors: Smit Eric , Leeflang Paula , Glandorf Boet , Dirk van Elsas Jan , Wernars Karel
Published: December 2019

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in
Escherichia coli
by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify
Basidiomycota
and
Zygomycota
, whereas EF4-fung5 amplified mainly
Ascomycota
. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.
Characterization of the Binding Protein-Dependent Cellobiose and Cellotriose Transport System of the Cellulose Degrader
Streptomyces reticuli
Authors: Schlösser Andreas , Jantos Jens , Hackmann Karl , Schrempf Hildgund
Published: December 2019

Streptomyces reticuli
has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH
2
-terminal part of an intracellular β-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the
ceb
operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in
S. reticuli
and
Streptomyces lividans
. Transcription of polycistronic
cebEFG
and
bglC
mRNAs is induced by cellobiose, whereas the
cebR
gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of
S. reticuli
, since insertional inactivation of the
cebE
gene prevents high-affinity uptake of cellobiose.
Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound
Authors: Li Kaichang , Xu Feng , Eriksson Karl-Erik L.
Published: December 2019

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order:Trametes villosalaccase (TvL) >Pycnoporus cinnabarinuslaccase (PcL) >Botrytis cinerealaccase (BcL) >Myceliophthora thermophilalaccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon bothkcatand the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affectkcatand the oxidation rate of dimer I.
Mapping of Sugar and Amino Acid Availability in Soil around Roots with Bacterial Sensors of Sucrose and Tryptophan
Authors: Jaeger C. H. , Lindow S. E. , Miller W. , Clark E. , Firestone M. K.
Published: December 2019

We developed a technique to map the availability of sugars and amino acids along live roots in an intact soil-root matrix with native microbial soil flora and fauna present. It will allow us to study interactions between root exudates and soil microorganisms at the fine spatial scale necessary to evaluate mechanisms of nitrogen cycling in the rhizosphere.
Erwinia herbicola
299R harboring a promoterless ice nucleation reporter gene, driven by either of two nutrient-responsive promoters, was used as a biosensor. Strain 299RTice exhibits tryptophan-dependent ice nucleation activity, while strain 299R(p61RYice) expresses ice nucleation activity proportional to sucrose concentration in its environment. Both biosensors exhibited up to 100-fold differences in ice nucleation activity in response to varying substrate abundance in culture. The biosensors were introduced into the rhizosphere of the annual grass
Avena barbata
and, as a control, into bulk soil. Neither strain exhibited significant ice nucleation activity in the bulk soil. Both tryptophan and sucrose were detected in the rhizosphere, but they showed different spatial patterns. Tryptophan was apparently most abundant in soil around roots 12 to 16 cm from the tip, while sucrose was most abundant in soil near the root tip. The largest numbers of bacteria (determined by acridine orange staining and direct microscopy) occurred near root sections with the highest apparent sucrose or tryptophan exudation. High sucrose availability at the root tip is consistent with leakage of photosynthate from immature, rapidly growing root tissues, while tryptophan loss from older root sections may result from lateral root perforation of the root epidermis.
Reduction of Technetium by
Desulfovibrio desulfuricans
: Biocatalyst Characterization and Use in a Flowthrough Bioreactor
Authors: Lloyd J. R. , Ridley J. , Khizniak T. , Lyalikova N. N. , Macaskie L. E.
Published: December 2019

Resting cells of
Desulfovibrio desulfuricans
coupled the oxidation of a range of electron donors to Tc(VII) reduction. The reduced technetium was precipitated as an insoluble low-valence oxide. The optimum electron donor for the biotransformation was hydrogen, although rapid rates of reduction were also supported when formate or pyruvate was supplied to the cells. Technetium reduction was less efficient when the growth substrates lactate and ethanol were supplied as electron donors, while glycerol, succinate, acetate, and methanol supported negligible reduction. Enzyme activity was stable for several weeks and was insensitive to oxygen. Transmission electron microscopy showed that the radionuclide was precipitated at the periphery of the cell. Cells poisoned with Cu(II), which is selective for periplasmic but not cytoplasmic hydrogenases, were unable to reduce Tc(VII), a result consistent with the involvement of a periplasmic hydrogenase in Tc(VII) reduction. Resting cells, immobilized in a flowthrough membrane bioreactor and supplied with Tc(VII)-supplemented solution, accumulated substantial quantities of the radionuclide when formate was supplied as the electron donor, indicating the potential of this organism as a biocatalyst to treat Tc-contaminated wastewaters.
The Quiescent-Cell Expression System for Protein Synthesis in
Escherichia coli
Authors: Rowe Duncan C. D. , Summers David K.
Published: December 2019

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in
Escherichia coli
. It is dependent on the controlled overexpression of a small RNA called Rcd in
hns
mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.
Modulation of Lipid Metabolism and Spiramycin Biosynthesis in
Streptomyces ambofaciens
Unstable Mutants
Authors: Schauner Catherine , Dary Annie , Lebrihi Ahmed , Leblond Pierre , Decaris Bernard , Germain Pierre
Published: December 2019

Streptomyces ambofaciens
is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (
orfPS
), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205
+
Spi
−
). The product of
orfPS
is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of
orfPS
also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205
−
Spi
+
). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.
Interactions of Saprophytic Yeasts with a
nor
Mutant of
Aspergillus flavus
Authors: Hua Sui-Sheng T. , Baker James L. , Flores-Espiritu Melanie
Published: December 2019

The
nor
mutant of
Aspergillus flavus
has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the
nor
mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the
nor
mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of
A. flavus
. These yeasts may be useful for reducing aflatoxin contamination of food commodities.
Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria
Authors: Lee Sang Yup , Choi Jong-il , Han Kyuboem , Song Ji Yong
Published: December 2019

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including
Ralstonia eutropha
,
Alcaligenes latus
, and recombinant
Escherichia coli
. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant
E. coli
, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30°C was higher than 10
4
EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant
E. coli
by simple NaOH digestion.