bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between Glycosyl Hydrolase Inventory and Growth Physiology of the Hyperthermophile
Pyrococcus furiosus
on Carbohydrate-Based Media
Authors: Driskill Lance E. , Kusy Kevin , Bauer Michael W. , Kelly Robert M.
Published: December 2019

Utilization of a range of carbohydrates for growth by the hyperthermophile
Pyrococcus furiosus
was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously,
P. furiosus
had been found to grow in batch cultures on several α-linked carbohydrates and cellobiose but not on glucose or other β-linked sugars. Although
P. furiosus
was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98°C occurred on β-1,3- and β-1,3–β-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from
P. furiosus
were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of β-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98°C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For
P. furiosus
, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism.
Growth Characteristics of
Heterosigma akashiwo
Virus and Its Possible Use as a Microbiological Agent for Red Tide Control
Authors: Nagasaki Keizo , Tarutani Kenji , Yamaguchi Mineo
Published: December 2019

The growth characteristics of
Heterosigma akashiwo
virus clone 01 (HaV01) were examined by performing a one-step growth experiment. The virus had a latent period of 30 to 33 h and a burst size of 7.7 × 10
2
lysis-causing units in an infected cell. Transmission electron microscopy showed that the virus particles formed on the peripheries of viroplasms, as observed in a natural
H. akashiwo
cell. Inoculation of HaV01 into a mixed algal culture containing four phytoplankton species,
H. akashiwo
H93616
,
Chattonella antiqua
(a member of the family Raphidophyceae),
Heterocapsa triquetra
(a member of the family Dinophyceae), and
Ditylum brightwellii
(a member of the family Bacillariophyceae), resulted in selective growth inhibition of
H. akashiwo
. Inoculation of HaV01 and
H. akashiwo
H93616
into a natural seawater sample produced similar results. However, a natural
H. akashiwo
red tide sample did not exhibit any conspicuous sensitivity to HaV01, presumably because of the great diversity of the host species with respect to virus infection. The growth characteristics of the lytic virus infecting the noxious harmful algal bloom-causing alga were considered, and the possibility of using this virus as a microbiological agent against
H. akashiwo
red tides is discussed.
Purification and Characterization of Gentisate 1,2-Dioxygenases from
Pseudomonas alcaligenes
NCIB 9867 and
Pseudomonas putida
NCIB 9869
Authors: Feng Yongmei , Khoo Hoon Eng , Poh Chit Laa
Published: December 2019

Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from
Pseudomonas alcaligenes
NCIB 9867 (P25X) and
Pseudomonas putida
NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1,2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1,2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent

K
m

s of 92 and 143 μM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with
k
cat
/

K
m

values of 44.08 × 10
4
s
−1
M
−1
for the P25X enzyme and 39.34 × 10
4
s
−1
M
−1
for the P35X enzyme. Higher
k
cat
/

K
m

values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1,2-dioxygenases was around 50°C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe
2+
but were completely inhibited by the presence of 5 mM Cu
2+
. Partial inhibition of both enzymes was also observed with 5 mM Mn
2+
, Zn
2+
, and EDTA.
Molecular Characterization of the Genes
pcaG
and
pcaH
, Encoding Protocatechuate 3,4-Dioxygenase, Which Are Essential for Vanillin Catabolism in
Pseudomonas
sp. Strain HR199
Authors: Overhage Jörg , Kresse Andreas U. , Priefert Horst , Sommer Horst , Krammer Gerhard , Rabenhorst Jürgen , Steinbüchel Alexander
Published: December 2019

Pseudomonas
sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a
Pseudomonas
sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp
Eco
RI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to
pcaG
and
pcaH
, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of
Agrobacterium tumefaciens
and PobR of
Pseudomonas aeruginosa
, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only
pcaH
, this mutant was most probably lacking a functional β subunit of the protocatechuate 3,4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the
ortho
cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a
syn
absolute stereochemical course, which is normally only observed for
cis
,
cis
-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the
ortho
-cleavage pathway in
Pseudomonas
sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.
Molecular Differentiation of
Renibacterium salmoninarum
Isolates from Worldwide Locations
Authors: Grayson Thomas H. , Cooper Lynne F. , Atienzar Franck A. , Knowles Mark R. , Gilpin Martyn L.
Published: December 2019

Renibacterium salmoninarum
is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of
R. salmoninarum
. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of
R. salmoninarum
is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of
R. salmoninarum
obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.
Effect of Phenylurea Herbicides on Soil Microbial Communities Estimated by Analysis of 16S rRNA Gene Fingerprints and Community-Level Physiological Profiles
Authors: el Fantroussi Saïd , Verschuere Laurent , Verstraete Willy , Top Eva M.
Published: December 2019

The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.
Anaerobic Oxidation of o -Xylene, m -Xylene, and Homologous Alkylbenzenes by New Types of Sulfate-Reducing Bacteria
Authors: Harms Gerda , Zengler Karsten , Rabus Ralf , Aeckersberg Frank , Minz Dror , Rosselló-Mora Ramon , Widdel Friedrich
Published: December 2019

Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with
o
-xylene and
m
-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the δ subclass of the class
Proteobacteria
. Strain oXyS1 showed the highest similarities to
Desulfobacterium cetonicum
and
Desulfosarcina variabilis
(similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being
Desulfococcus multivorans
(similarity value, 86.9%). Complete mineralization of
o
-xylene and
m
-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene,
o
-ethyltoluene, benzoate, and
o
-methylbenzoate in addition to
o
-xylene. Strain mXyS1 oxidized toluene,
m
-ethyltoluene,
m
-isoproyltoluene, benzoate, and
m
-methylbenzoate in addition to
m
-xylene. Strain oXyS1 did not utilize
m
-alkyltoluenes, whereas strain mXyS1 did not utilize
o
-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide.
Fungal Degradation of Lipophilic Extractives in
Eucalyptus globulus
Wood
Authors: Gutiérrez Ana , del Río José C. , Martínez María Jesús , Martínez Angel T.
Published: December 2019

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl
2
-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way,
Phlebia radiata
,
Funalia trogii
,
Bjerkandera adusta
, and
Poria subvermispora
strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives.
Ophiostoma piliferum
, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.
Homofermentative Production of
d
- or
l
-Lactate in Metabolically Engineered
Escherichia coli
RR1
Authors: Chang Dong-Eun , Jung Heung-Chae , Rhee Joon-Shick , Pan Jae-Gu
Published: December 2019

We investigated metabolic engineering of fermentation pathways in
Escherichia coli
for production of optically pure
d
- or
l
-lactate. Several
pta
mutant strains were examined, and a
pta
mutant of
E. coli
RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to
d
-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in
ppc
made the mutant produce
d
-lactate like a homofermentative lactic acid bacterium. When the
pta ppc
double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of
d
-lactate production, more than 62.2 g of
d
-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous
l
-lactate pathway, an
l
-lactate dehydrogenase gene from
Lactobacillus casei
was introduced into a
pta ldhA
strain which lacked phosphotransacetylase and
d
-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to
l
-lactate as the major fermentation product, and up to 45 g of
l
-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of
E. coli
can be reoriented to the production of
d
-lactate, an indigenous fermentation product, or to the production of
l
-lactate, a nonindigenous fermentation product.
Role of Calcium in Activity and Stability of the
Lactococcus lactis
Cell Envelope Proteinase
Authors: Exterkate Fred A. , Alting Arno C.
Published: December 2019

The mature lactococcal cell envelope proteinase (CEP) consists of an N-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. Different types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca
2+
from the native cell-bound CEP of
Lactococcus lactis
SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25°C (pH 6.5) can be measured. The consequences of Ca
2+
removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). Autoproteolytic release of CEP from cells concerns this so-called “Ca-free” form only and occurs most efficiently in the case of the Wg2 CEP. The results of a study of the relationship between the Ca
2+
concentration and the stability and activity of the cell-bound SK11 CEP at 25°C suggested that binding of at least two Ca
2+
ions occurred. Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca
2+
dependence of the cell-bound CEP. The results are discussed in terms of predicted Ca
2+
binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site. We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.
Location and Survival of Leaf-Associated Bacteria in Relation to Pathogenicity and Potential for Growth within the Leaf
Authors: Wilson M. , Hirano S. S. , Lindow S. E.
Published: December 2019

The growth and survival of pathogenic and nonpathogenic
Pseudomonas syringae
strains and of the nonpathogenic species
Pantoea agglomerans
,
Stenotrophomonas maltophilia
, and
Methylobacterium organophilum
were compared in the phyllosphere of bean. In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress. The sizes of the total leaf-associated populations of the pathogenic
P. syringae
strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions. In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in “protected sites” that were inaccessible to these agents. In general, the population sizes in protected sites increased with time after inoculation of plants. The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions. When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed “internal” populations were greater for pathogenic
P. syringae
strains than for nonpathogenic
P. syringae
strains. The sizes of the populations of the nonpathogenic species failed to increase or even decreased. The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation. While the sizes of the populations of the pathogenic
P. syringae
strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of
P. syringae
,
P. agglomerans
, and
S. maltophilia
decreased when the organisms were applied to plants. The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration. Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves.
Detection of Cytolethal Distending Toxin Activity and
cdt
Genes in
Campylobacter
spp. Isolated from Chicken Carcasses
Authors: Eyigor Aysegul , Dawson Karl A. , Langlois Bruce E. , Pickett Carol L.
Published: December 2019

This study was designed to determine whether isolates from chicken carcasses, the primary source of
Campylobacter jejuni
and
Campylobacter coli
in human infections, commonly carry the
cdt
genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates.
Campylobacter
spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets.
Campylobacter
spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as
C. jejuni
, and 35 (33%) were identified as
C. coli
. PCR tests amplified portions of the
cdt
genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the
C. jejuni
and
C. coli cdt
genes, but that the restriction patterns of the
cdt
genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all
C. jejuni
strains except four (94%) had mean CDT titers greater than 100. Only one
C. jejuni
strain appeared to produce no active CDT.
C. coli
isolates produced little or no toxin. These results confirm the high rate of
Campylobacter
sp. contamination of fresh chicken carcasses and indicate that
cdt
genes may be universally present in
C. jejuni
and
C. coli
isolates from chicken carcasses.
PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria
Authors: Hallin Sara , Lindgren Per-Eric
Published: December 2019

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnirgenes were also established. Thecd1primers were designed to amplify a 778 to 799-bp region ofcd1-nirin the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nirsequences. Together, the two sets of PCR primers amplifiednirgenes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, butcd1-nirfragments were only obtained in five samples. PCR products of the expected sizes were verified asnirgenes after hybridization to DNA probes, except in one case. The sequencednirfragments were related to othernirsequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nirwere conserved, while broad-range primers targeting conserved regions ofcd1-nirseem to be difficult to find. We also report on the existence of Cu-nirinParacoccus denitrificansPd1222.
Molecular Analysis of Bacterial Community Structure and Diversity in Unimproved and Improved Upland Grass Pastures
Authors: McCaig Allison E. , Glover L. Anne , Prosser James I.
Published: December 2019

Bacterial community structure and diversity in rhizospheres in two types of grassland, distinguished by both plant species and fertilization regimen, were assessed by performing a 16S ribosomal DNA (rDNA) sequence analysis of DNAs extracted from triplicate soil plots. PCR products were cloned, and 45 to 48 clones from each of the six libraries were partially sequenced. Phylogenetic analysis of the resultant 275 clone sequences indicated that there was considerable variation in abundance in replicate unfertilized, unimproved soil samples and fertilized, improved soil samples but that there were no significant differences in the abundance of any phylogenetic group. Several clone sequences were identical in the 16S rDNA region analyzed, and the clones comprised eight pairs of duplicate clones and two sets of triplicate clones. Many clones were found to be most closely related to environmental clones obtained in other studies, although three clones were found to be identical to culturable species in databases. The clones were clustered into operational taxonomic units at a level of sequence similarity of >97% in order to quantify diversity. In all, 34 clusters containing two or more sequences were identified, and the largest group contained nine clones. A number of diversity, dominance, and evenness indices were calculated, and they all indicated that diversity was high, reflecting the low coverage of rDNA libraries achieved. Differences in diversity between sample types were not observed. Collector’s curves, however, indicated that there were differences in the underlying community structures; in particular, there was reduced diversity of organisms of the α subdivision of the class
Proteobacteria
(α-proteobacteria) in improved soils.
Effects of Nickel and Cobalt on Kinetics of Methanol Conversion by Methanogenic Sludge as Assessed by On-Line CH
4
Monitoring
Authors: Gonzalez-Gil Graciela , Kleerebezem Robbert , Lettinga Gatze
Published: December 2019

When metals were added in a pulse mode to methylotrophic-methanogenic biomass, three methane production rate phases were recognized. Increased concentrations of Ni and Co accelerated the initial exponential and final arithmetic increases in the methane production rate and reduced the temporary decrease in the rate. When Ni and Co were added continuously, the temporary decrease phase was eliminated and the exponential production rate increased. We hypothesize that the temporary decrease in the methane production rate and the final arithmetic increase in the methane production rate were due to micronutrient limitations and that the precipitation-dissolution kinetics of metal sulfides may play a key role in the biovailability of these compounds.
Production of Manganese Peroxidase and Organic Acids and Mineralization of
14
C-Labelled Lignin (
14
C-DHP) during Solid-State Fermentation of Wheat Straw with the White Rot Fungus
Nematoloma frowardii
Authors: Hofrichter Martin , Vares Tamara , Kalsi Mika , Galkin Sari , Scheibner Katrin , Fritsche Wolfgang , Hatakka Annele
Published: December 2019

The basidiomycetous fungus
Nematoloma frowardii
produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic
14
C-ring-labelled lignin (
14
C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as
14
CO
2
, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the
14
C-DHP to
14
CO
2
(which accounted for up to 8% of the initial radioactivity added) and
14
C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).
Construction and Analysis of a Library for Random Insertional Mutagenesis in
Streptococcus pneumoniae
: Use for Recovery of Mutants Defective in Genetic Transformation and for Identification of Essential Genes
Authors: Lee Myeong S. , Dougherty Brian A. , Madeo Anne C. , Morrison Donald A.
Published: December 2019

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of
Streptococcus pneumoniae
, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in
Escherichia coli
. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the
Bacillus subtilis
competence gene
comFA
. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless
lacZ
reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.
Potential of Conjugal Transfer as a Strategy for the Introduction of Recombinant Genetic Material into Strains of
Lactobacillus helveticus
Authors: Thompson J. K. , McConville K. J. , McReynolds C. , Collins M. A.
Published: December 2019

Cointegrates generated between a plasmid pIP501 deletion derivative (pVA797) and nonconjugative shuttle vector pSA3 were confirmed as capable of exconjugation from lactococci into a range of strains of
Lactobacillus helveticus
with the concomitant expression of a recombinant gene. The plasmid cointegrate that was formed appeared to be segregationally stable at 37°C in some host strains. In all strains, however, the plasmid became increasingly unstable as the incubation temperature was raised. The technique offers not only a generalized method for the introduction of novel genetic material into this important industrial microbe but also the possibility of exploiting the thermal sensitivity of the plasmid to enable it to act as a delivery system for the integration of cloned genes into the bacterial chromosome, at restrictive temperatures, by recombination at regions of homology.
Expression of an
Aspergillus niger
Phytase Gene (
phyA
) in
Saccharomyces cerevisiae
Authors: Han Yanming , Wilson David B. , Lei Xin gen
Published: December 2019

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an
Aspergillus niger
phytase gene (
phyA
) in
Saccharomyces cerevisiae
and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the
phyA
gene was inserted into the expression vector pYES2 and was expressed in
S. cerevisiae
as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the
phyA
gene was expressed as an active, extracellular phytase in
S. cerevisiae
, and its thermostability was affected by glycosylation.
Selective Isolation and Distribution of
Sporichthya
Strains in Soil
Authors: Suzuki Shin-ichi , Okuda Toru , Komatsubara Saburo
Published: December 2019

A simplified enrichment method in which centrifugation is used for selective isolation of
Sporichthya
strains from soil is described. Gellan gum plus 2 mM CaCl
2
stimulated growth of
Sporichthya polymorpha
KCC A0089 so that this organism was readily recognized on an isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27°C for 60 min and centrifuging it at 1,000 ×
g
for 10 min. Dry heat treatment at 80°C for 60 min increased the ratio of
Sporichthya
colonies to nonfilamentous bacteria on a gellan gum plate. Since
S. polymorpha
was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating
Sporichthya
strains. Our isolates were identified as
Sporichthya
strains on the basis of their morphological and chemotaxonomic characteristics. By combining the techniques described above, we isolated a number of
Sporichthya
strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States.
Sporichthya
strains are widely distributed in the world. To our knowledge, this is the first time that
Sporichthya
strains have been isolated from locations other than the United States or Japan.
Molecular Analyses of the Methane-Oxidizing Microbial Community in Rice Field Soil by Targeting the Genes of the 16S rRNA, Particulate Methane Monooxygenase, and Methanol Dehydrogenase
Authors: Henckel Thilo , Friedrich Michael , Conrad Ralf
Published: December 2019

Rice field soil with a nonsaturated water content induced CH
4
consumption activity when it was supplemented with 5% CH
4
. After a lag phase of 3 days, CH
4
was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH
4
mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the γ subdivision of the class
Proteobacteria
[γ-
Proteobacteria
]) and type II methylotrophs (members of the α-
Proteobacteria
) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (
pmoA
) and methanol dehydrogenase (
mxaF
), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH
4
was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera
Flavobacterium
and
Bacillus
and was not influenced by CH
4
. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera
Methylobacter
,
Methylococcus
, and
Methylocystis
. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the
mxaF
and
pmoA
genes revealed that there were pronounced community shifts when CH
4
oxidation began. High CH
4
concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.
Accumulation of Trehalose by Overexpression of
tps1
, Coding for Trehalose-6-Phosphate Synthase, Causes Increased Resistance to Multiple Stresses in the Fission Yeast
Schizosaccharomyces pombe
Authors: Soto Teresa , Fernández Juana , Vicente-Soler Jero , Cansado Jose , Gacto Mariano
Published: December 2019

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast
Schizosaccharomyces pombe
. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of
S. pombe
transformed with a plasmid bearing the
tps1
gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in
S. pombe
.
Biodiversity of
Clostridium botulinum
Type E Strains Isolated from Fish and Fishery Products
Authors: Hyytiä Eija , Hielm Sebastian , Björkroth Johanna , Korkeala Hannu
Published: December 2019

The genetic biodiversity of
Clostridium botulinum
type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (
Sma
I-
Xma
I and
Xho
I) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.
Molecular Analysis of Microbial Community Structures in Pristine and Contaminated Aquifers: Field and Laboratory Microcosm Experiments
Authors: Shi Y. , Zwolinski M. D. , Schreiber M. E. , Bahr J. M. , Sewell G. W. , Hickey W. J.
Published: December 2019

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly
Bacteria
;
Eucarya
and
Archaea
were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% β- and γ-
Proteobacteria
(B+G), 31 to 35% α-
Proteobacteria
(ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly
Bacteria
(83 to 101%) and lacked detectable
Archaea
but differed in that a small fraction (2 to 8%) of
Eucarya
was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the
Bacteria
community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.
Anaerobic Dehalogenation of Hydroxylated Polychlorinated Biphenyls by
Desulfitobacterium dehalogenans
Authors: Wiegel Juergen , Zhang Xiaoming , Wu Qingzhong
Published: December 2019

Ten years after reports on the existence of anaerobic dehalogenation of polychlorinated biphenyls (PCBs) in sediment slurries, we report here on the rapid reductive dehalogenation of
para
-hydroxylated PCBs (HO-PCBs), the excreted main metabolites of PCB in mammals, which can exhibit estrogenic and antiestrogenic activities in humans. The anaerobic bacterium
Desulfitobacterium dehalogenans
completely dehalogenates all flanking chlorines (chlorines in
ortho
position to the
para
-hydroxyl group) from congeners such as 3,3′,5,5′-tetrachloro-4,4′-dihydroxybiphenyl.
Microbial Degradation of Octamethylcyclotetrasiloxane
Authors: Grümping R. , Michalke K. , Hirner A. V. , Hensel R.
Published: December 2019

The microbial degradation of low-molecular-weight polydimethylsiloxanes was investigated through laboratory experiments. Octamethylcyclotetrasiloxane was found to be biodegraded under anaerobic conditions in composted sewage sludge, as monitored by the occurrence of the main polydimethylsiloxane degradation product, dimethylsilanediol, compared to that found in experiments with sterilized control samples.
High-Resolution Genotyping of
Campylobacter
Strains Isolated from Poultry and Humans with Amplified Fragment Length Polymorphism Fingerprinting
Authors: Duim Birgitta , Wassenaar Trudy M. , Rigter Alan , Wagenaar Jaap
Published: December 2019

For epidemiological studies of
Campylobacter
infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of
Campylobacter jejuni
and
Campylobacter coli
strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases
Hin
dIII and
Hha
I were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a
C. jejuni
strain, an isogenic flagellin mutant, and distinct
C. jejuni
strains having known pulsed-field gel electrophoresis and
fla
PCR-restriction fragment length polymorphism genotypes. Unrelated
C. jejuni
strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five
Campylobacter
strains obtained from poultry farms in The Netherlands grouped in three
C. jejuni
clusters that were separate from a
C. coli
cluster. The band patterns of 10
C. jejuni
strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of
Campylobacter
species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of
Campylobacter
strains and is extremely useful for epidemiological investigations.