bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Diversity of
nifH
Gene Sequences in
Paenibacillus azotofixans
Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments
Authors: Rosado Alexandre S. , Duarte Gabriela F. , Seldin Lucy , Van Elsas Jan Dirk
Published: December 2019

The diversity of dinitrogenase reductase gene (
nifH
) fragments in
Paenibacillus azotofixans
strains was investigated by using molecular methods. The partial
nifH
gene sequences of eight
P. azotofixans
strains, as well as one strain each of the close relatives
Paenibacillus durum
,
Paenibacillus polymyxa
, and
Paenibacillus macerans
, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two
nifH
sequence clusters, designated clusters I and II, in
P. azotofixans
. The data further indicated that there was sequence divergence among the
nifH
genes of
P. azotofixans
strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all
nifH
cluster II sequences were similar to the alternative (
anf
) nitrogenase sequence. A nested PCR assay for the detection of
nifH
(cluster I) of
P. azotofixans
was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the
P. azotofixans nifH
gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of
nifH
in
P. azotofixans
strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different
nifH
genes. Moreover,
nifH
is probably a multicopy gene in
P. azotofixans
. Both similarities and differences were detected in the
P. azotofixans nifH
DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.
Acetoacetyl Coenzyme A Reductase and Polyhydroxybutyrate Synthesis in
Rhizobium
(
Cicer
) sp. Strain CC 1192
Authors: Chohan Shahid N. , Copeland Les
Published: December 2019

Biochemical controls that regulate the biosynthesis of poly-3-hydroxybutyrate (PHB) were investigated in
Rhizobium
(
Cicer
) sp. strain CC 1192. This species is of interest for studying PHB synthesis because the polymer accumulates to a large extent in free-living cells but not in bacteroids during nitrogen-fixing symbiosis with chickpea (
Cicer arietinum
L.) plants. Evidence is presented that indicates that CC 1192 cells retain the enzymic capacity to synthesize PHB when they differentiate from the free-living state to the bacteroid state. This evidence includes the incorporation by CC 1192 bacteroids of radiolabel from [
14
C]malate into 3-hydroxybutyrate which was derived by chemically degrading insoluble material from bacteroid pellets. Furthermore, the presence of an NADPH-dependent acetoacetyl coenzyme A (CoA) reductase, which was specific for
R
-(−)-3-hydroxybutyryl-CoA and NADP
+
in the oxidative direction, was demonstrated in extracts from free-living and bacteroid cells of CC 1192. Activity of this enzyme in the reductive direction appeared to be regulated at the biochemical level mainly by the availability of substrates. The CC 1192 cells also contained an NADH-specific acetoacetyl-CoA reductase which oxidized
S
-(+)-3-hydroxybutyryl-CoA. A membrane preparation from CC 1192 bacteroids readily oxidized NADH but not NADPH, which is suggested to be a major source of reductant for nitrogenase. Thus, a high ratio of NADPH to NADP
+
, which could enhance delivery of reductant to nitrogenase, could also favor the reduction of acetoacetyl-CoA for PHB synthesis. This would mean that fine controls that regulate the partitioning of acetyl-CoA between citrate synthase and 3-ketothiolase are important in determining whether PHB accumulates.
Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by
Bacillus subtilis
Authors: Stephenson Keith , Harwood Colin R.
Published: December 2019

AmyL, an extracellular α-amylase from
Bacillus licheniformis
, is resistant to extracellular proteases secreted by
Bacillus subtilis
during growth. Nevertheless, when AmyL is produced and secreted by
B. subtilis
, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the
B. subtilis wprA
gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible
wprA
gene, we show that a product of
wprA
, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which
wprA
is not expressed exhibits an increased yield of α-amylase. The potential role of
wprA
in protein secretion is discussed, together with implications for the use of
B. subtilis
and related bacteria as hosts for the secretion of heterologous proteins.
A Novel β-
N
-Acetylglucosaminidase from
Streptomyces thermoviolaceus
OPC-520: Gene Cloning, Expression, and Assignment to Family 3 of the Glycosyl Hydrolases
Authors: Tsujibo Hiroshi , Hatano Naoya , Mikami Tadahisa , Hirasawa Ayako , Miyamoto Katsushiro , Inamori Yoshihiko
Published: December 2019

A β-
N
-acetylglucosaminidase gene (
nagA
) of
Streptomyces thermoviolaceus
OPC-520 was cloned in
Streptomyces lividans
66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an
M
r
of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-
N
-acetylglucosaminidase expressed by
S. lividans
. The enzyme shares no sequence similarity with the classical β-
N
-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from
S. thermoviolaceus
OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent

K
m

and
V
max
values for
p
-nitrophenyl-β-
N
-acetylglucosamine were 425.7 μM and 24.8 μmol min
−1
mg of protein
−1
, respectively.
Sulfate-Reducing Bacteria and Their Activities in Cyanobacterial Mats of Solar Lake (Sinai, Egypt)
Authors: Teske Andreas , Ramsing Niels B. , Habicht Kirsten , Fukui Manabu , Küver Jan , Jørgensen Bo Barker , Cohen Yehuda
Published: December 2019

The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 10
6
and 10
7
cultivable sulfate-reducing bacteria ml
−1
and showed sulfate reduction rates between 1,000 and 2,200 nmol ml
−1
day
−1
, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing
Desulfonema
bacteria were found in variable densities of 10
4
to 10
6
cells ml
−1
. A
Desulfonema
-related, diurnally migrating bacterium was detected with PCR and denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesis rates showed that CO
2
from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO
2
demand of the mat.
Microbial Dechlorination of 2,3,5,6-Tetrachlorobiphenyl under Anaerobic Conditions in the Absence of Soil or Sediment
Authors: Cutter Leah , Sowers Kevin R. , May Harold D.
Published: December 2019

Bacterial enrichment cultures developed with Baltimore Harbor (BH) sediments were found to reductively dechlorinate 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) when incubated in a minimal estuarine medium containing short-chain fatty acids under anaerobic conditions with and without the addition of sediment. Primary enrichment cultures formed both
meta
and
ortho
dechlorination products from 2,3,5,6-CB. The lag time preceding dechlorination decreased from 30 to less than 20 days as the cultures were sequentially transferred into estuarine medium containing dried, sterile BH sediment. In addition, only
ortho
dechlorination was observed following transfer of the cultures. Sequential transfer into medium without added sediment also resulted in the development of a strict
ortho
-dechlorinating culture following a lag of more than 100 days. Upon further transfer into the minimal medium without sediment, the lag time decreased to less than 50 days. At this stage all cultures, regardless of the presence of sediment, would produce 2,3,5-CB and 3,5-CB from 2,3,5,6-CB. The strict
ortho
-dechlorinating activity in the sediment-free cultures has remained stable for more than 1 year through several transfers. These results reveal that the classical microbial enrichment technique using a minimal medium with a single polychlorinated biphenyl (PCB) congener selected for
ortho
dechlorination of 2,3,5,6-CB. Furthermore, this is the first report of sustained anaerobic PCB dechlorination in the complete absence of soil or sediment.
Phage Display of a Biologically Active
Bacillus thuringiensis
Toxin
Authors: Kasman Laura M. , Lukowiak Andrew A. , Garczynski Stephen F. , McNall Rebecca J. , Youngman Phil , Adang Michael J.
Published: December 2019

Activated forms of
Bacillus thuringiensis
insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in
Escherichia coli
. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in
E. coli
, since the molecular biology tools available for
Bacillus
are limited. To this end, we show that activated
B. thuringiensis
toxin (Cry1Ac) can be expressed in
E. coli
as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of
B. thuringiensis
toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.
Whole-Cell Immunolocalization of Nitrogenase in Marine Diazotrophic Cyanobacteria,
Trichodesmium
spp
Authors: Lin Senjie , Henze Sheri , Lundgren Pernilla , Bergman Birgitta , Carpenter Edward J.
Published: December 2019

The mechanism by which planktonic marine cyanobacteria of the genus
Trichodesmium
fix N
2
aerobically during photosynthesis without heterocysts is unknown. As an aid in understanding how these species protect nitrogenase, we have developed an immunofluorescence technique coupled to light microscopy (IF-LM) with which intact cyanobacteria can be immunolabeled and the distribution patterns of nitrogenase and other proteins can be described and semiquantified. Chilled ethanol was used to fix the cells, which were subsequently made permeable to antibodies by using dimethyl sulfoxide. Use of this technique demonstrated that about 3 to 20 cells (mean ± standard deviation, 9 ± 4) consecutively arranged in a
Trichodesmium
trichome were labeled with the nitrogenase antibody. The nitrogenase-containing cells were distributed more frequently around the center of the trichome and were rarely found at the ends. On average 15% of over 300 randomly encountered cells examined contained nitrogenase. The percentage of nitrogenase-containing cells (nitrogenase index [NI]) in an exponential culture was higher early in the light period than during the rest of the light-dark cycle, while that for a stationary culture was somewhat constant at a lower level throughout the light-dark cycle. The NI was not affected by treatment of the cultures with the photosynthetic inhibitor dichloro 1,3′-dimethyl urea or with low concentrations of ammonium (NH
4
Cl). However, incubation of cultures with 0.5 μM NH
4
Cl over 2 days reduced the NI. The IF technique combined with
14
C autoradiography showed that the CO
2
fixation rate was lower in nitrogenase-containing cells. The results of the present study suggest that (i) the IF-LM technique may be a useful tool for in situ protein localization in cyanobacteria, (ii) cell differentiation occurs in
Trichodesmium
and only a small fraction of cells in a colony have the potential to fix nitrogen, (iii) the photosynthetic activity (CO
2
uptake) is reduced if not absent in N
2
-fixing cells, and (iv) variation in the NI may be a modulator of nitrogen-fixing activity.
Minimally Invasive Detection of
Piscirickettsia salmonis
in Cultivated Salmonids via the PCR
Authors: Marshall Sergio , Heath Sekou , Henríquez Vitalia , Orrego Cristián
Published: December 2019

The attributes of the PCR allowed implementation of an assay for specific detection of
Piscirickettsia salmonis
from a few microliters of fish serum. This opens the way to less invasive modes of sampling for this microbial pathogen in salmonids.
Failure of
Ixodes
Ticks To Inherit
Borrelia afzelii
Infection
Authors: Matuschka Franz-Rainer , Schinkel Thomas W. , Klug Birte , Spielman Andrew , Richter Dania
Published: December 2019

To define conditions promoting inherited infection by Lyme disease spirochetes in
Ixodes
ticks, we variously infected ticks with
Borrelia afzelii
and examined their progenies by dark-field microscopy, immunofluorescence, PCR, and serial passage. No episode of inherited infection was evident, regardless of instar or gender infected or frequency of exposure. We suggest that these spirochetes rarely, if ever, are inherited by vector ticks.
Cloning and Expression of the
Listeria monocytogenes
Scott A
ptsH
and
ptsI
Genes, Coding for HPr and Enzyme I, Respectively, of the Phosphotransferase System
Authors: Christensen Douglas P. , Benson Andrew K. , Hutkins Robert W.
Published: December 2019

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different carbohydrates in bacteria. In order to examine the role of the PTS in the physiology of
Listeria monocytogenes
, we identified the
ptsH
and
ptsI
genes encoding the HPr and enzyme I proteins, respectively, of the PTS. Nucleotide sequence analysis indicated that the predicted proteins are nearly 70% similar to HPr and enzyme I proteins from other organisms. Purified
L. monocytogenes
HPr overexpressed in
Escherichia coli
was also capable of complementing an HPr defect in heterologous extracts of
Staphylococcus aureus ptsH
mutants. Additional studies of the transcriptional organization and control indicated that the
ptsH
and
ptsI
genes are organized into a transcription unit that is under the control of a consensus-like promoter and that expression of these genes is mediated by glucose availability and pH or by by-products of glucose metabolism.
Modeling of the Competitive Growth of
Listeria monocytogenes
and
Lactococcus lactis
in Vegetable Broth
Authors: Breidt Frederick , Fleming Henry P.
Published: December 2019

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of
Lactococcus lactis
and
Listeria monocytogenes
were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of
L. monocytogenes
in competition with a strain of
L. lactis
which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods.
Analysis ofEscherichia coliO157:H7 Survival in Ovine or Bovine Manure and Manure Slurry
Authors: Kudva Indira T. , Blanch Kathryn , Hovde Carolyn J.
Published: December 2019

Farm animal manure or manure slurry may disseminate, transmit, or propagateEscherichia coliO157:H7. In this study, the survival and growth ofE. coliO157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined. A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions.E. coliO157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <102to 106CFU/g at different times over the course of the experiment. The DNA fingerprints ofE. coliO157:H7 isolated at month 1 and month 12 were identical or very similar. A secondE. coliO157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months. AnE. coliO157:H7-positive bovine manure pile was culture positive for 47 days. In the laboratory,E. coliO157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at −20, 4, 23, 37, 45, and 70°C.E. coliO157:H7 survived best in manure incubated without aeration at temperatures below 23°C, but it usually survived for shorter periods of time than it survived in manure held in the environment. The bacterium survived at least 100 days in bovine manure frozen at −20°C or in ovine manure incubated at 4 or 10°C for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days. In addition, we found that the Shiga toxin type 1 and 2 genes inE. coliO157:H7 had little or no influence on bacterial survival in manure or manure slurry. The long-term survival ofE. coliO157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions.
Role of Endoproteolytic Dibasic Proprotein Processing in Maturation of Secretory Proteins in
Trichoderma reesei
Authors: Goller Sabine P. , Schoisswohl Doris , Baron Michel , Parriche Martine , Kubicek Christian P.
Published: December 2019

Cell extracts of
Trichoderma reesei
exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca
2+
ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM
p
-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by
T. reesei
. Secretion of xylanase I (proprotein processing sequence -R-R-↓-R-↓-A-) and xylanase II (-K-R-↓-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R-↓-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A-↓-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from
Streptoalloteichus hindustanus
, fused to a series of model proprotein-processing sequences downstream of the expression signals of the
Aspergillus nidulans gpdA
promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in
T. reesei
and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.
Competitive Dominance among Strains of Luminous Bacteria Provides an Unusual Form of Evidence for Parallel Evolution in Sepiolid Squid-Vibrio Symbioses
Authors: Nishiguchi Michele K. , Ruby Edward G. , McFall-Ngai Margaret J.
Published: December 2019

One of the principal assumptions in symbiosis research is that associated partners have evolved in parallel. We report here experimental evidence for parallel speciation patterns among several partners of the sepiolid squid-luminous bacterial symbioses. Molecular phylogenies for 14 species of host squids were derived from sequences of both the nuclear internal transcribed spacer region and the mitochondrial cytochrome oxidase subunit I; the glyceraldehyde phosphate dehydrogenase locus was sequenced for phylogenetic determinations of 7 strains of bacterial symbionts. Comparisons of trees constructed for each of the three loci revealed a parallel phylogeny between the sepiolids and their respective symbionts. Because both the squids and their bacterial partners can be easily cultured independently in the laboratory, we were able to couple these phylogenetic analyses with experiments to examine the ability of the different symbiont strains to compete with each other during the colonization of one of the host species. Our results not only indicate a pronounced dominance of native symbiont strains over nonnative strains, but also reveal a hierarchy of symbiont competency that reflects the phylogenetic relationships of the partners. For the first time, molecular systematics has been coupled with experimental colonization assays to provide evidence for the existence of parallel speciation among a set of animal-bacterial associations.
Comparative Studies of Class IIa Bacteriocins of Lactic Acid Bacteria
Authors: Eijsink Vincent G. H. , Skeie Marianne , Middelhoven P. Hans , Brurberg May Bente , Nes Ingolf F.
Published: December 2019

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen
Listeria monocytogenes
was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibiting
Listeria
, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active toward
Listeria
but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria.
Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic
Bacillus
Isolate and Entire Nucleotide and Amino Acid Sequences
Authors: Igarashi Kazuaki , Hatada Yuji , Hagihara Hiroshi , Saeki Katsuhisa , Takaiwa Mikio , Uemura Takaaki , Ara Katsutoshi , Ozaki Katsuya , Kawai Shuji , Kobayashi Tohru , Ito Susumu
Published: December 2019

A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic
Bacillus
isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein
−1
, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable
Bacillus licheniformis
enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from
B. licheniformis
(68.9%),
Bacillus amyloliquefaciens
(66.7%), and
Bacillus stearothermophilus
(68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)
8
barrel motif in domain A.
Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-
ortho
-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA
Authors: Pulliam Holoman Tracey R. , Elberson Margaret A. , Cutter Leah A. , May Harold D. , Sowers Kevin R.
Published: December 2019

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that
ortho
dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a
Clostridium
sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class
Proteobacteria
, the low-G+C gram-positive subgroup, the
Thermotogales
subgroup, and a single species with sequence similarity to the deeply branching species
Dehalococcoides ethenogenes
were more predominant during active dechlorination of the PCB. Species with high sequence similarities to
Methanomicrobiales
and
Methanosarcinales
archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for
ortho
dechlorination of the congener. Deletion of
Clostridium
spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class
Bacteria
. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating
Dehalococcoides
, the 16S rDNA sequences associated with PCB
ortho
dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and
Thermotogales
subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.
Viral Pollution in the Environment and in Shellfish: Human Adenovirus Detection by PCR as an Index of Human Viruses
Authors: Pina Sonia , Puig Montserrat , Lucena Francisco , Jofre Joan , Girones Rosina
Published: December 2019

A study of the presence of human viruses (adenoviruses, enteroviruses, and hepatitis A viruses [HAVs]) in environmental and shellfish samples was carried out by applying DNA and cDNA amplification techniques by PCR. The detection of human adenoviruses by PCR was also examined as a potential molecular test to monitor viral pollution. The samples studied were urban and slaughterhouse sewage, river water, seawater, and shellfish. Enteroviruses were quantified by PFU in Buffalo green monkey kidney cells and fecal coliforms and phages of
Bacteroides fragilis
HSP40 were also evaluated in some of the samples. The amplification of viral DNA and cDNA has shown a high prevalence of human viruses that would not be detected by the use of classical techniques, such as the quantification of PFU in cell lines. The results of the analysis of slaughterhouse sewage samples together with the test of farm animal feces indicate that the adenoviruses and the HAVs detected in the environment are mostly of human origin. A significative correlation between the detection of human viruses by PCR and the values of bacteriophages of
B. fragilis
HSP40 in urban raw sewage was observed. Human adenoviruses were the viruses most frequently detected throughout the year, and all the samples that were positive for enteroviruses or HAVs were also positive for human adenoviruses. The results suggest that the detection of adenoviruses by PCR could be used as an index of the presence of human viruses in the environment where a molecular index is acceptable.
Mercury Resistance Is Encoded by Transferable Giant Linear Plasmids in Two Chesapeake Bay
Streptomyces
Strains
Authors: Ravel Jacques , Schrempf Hildgund , Hill Russell T.
Published: December 2019

The
Streptomyces
strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal
Streptomyces lividans
66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant
S. lividans
TK24 strain lacking plasmids and carrying a chromosomal deletion of the
mer
operon.
S. lividans
TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of
Streptomyces
strains CHR3 and CHR28.
Antibiotic Resistance in
Acinetobacter
spp. Isolated from Sewers Receiving Waste Effluent from a Hospital and a Pharmaceutical Plant
Authors: Guardabassi Luca , Petersen Andreas , Olsen John E. , Dalsgaard Anders
Published: December 2019

The possible increase of antibiotic-resistant bacteria in sewage associated with the discharge of wastewater from a hospital and a pharmaceutical plant was investigated by using
Acinetobacter
species as environmental bacterial indicators. The level of susceptibility to six antimicrobial agents was determined in 385
Acinetobacter
strains isolated from samples collected upstream and downstream from the discharge points of the hospital and the pharmaceutical plant. Results indicated that while the hospital waste effluent affected only the prevalence of oxytetracycline resistance, the discharge of wastewater from the pharmaceutical plant was associated with an increase in the prevalence of both single- and multiple-antibiotic resistance among
Acinetobacter
species in the sewers.
Influence of Growth Mode and Sucrose on Susceptibility of
Streptococcus sanguis
to Amine Fluorides and Amine Fluoride-Inorganic Fluoride Combinations
Authors: Embleton J. V. , Newman H. N. , Wilson M.
Published: December 2019

This study evaluated the susceptibility to amine fluorides (AmFs) of planktonic and biofilm cultures of
Streptococcus sanguis
grown with and without sucrose. Cultures were incubated with AmFs (250 mg of fluoride liter
−1
) for 1 min. The susceptibility of biofilms was less than that of the planktonic form and was further decreased by growth in the presence of sucrose.
Influence of Lipid Composition on Pediocin PA-1 Binding to Phospholipid Vesicles
Authors: Chen Yuhuan , Ludescher Richard D. , Montville Thomas J.
Published: December 2019

Pediocin PA-1 bound to anionic lipid vesicles with saturated or unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of the vesicles gave a higher relative dissociation constant for the peptide-lipid interactions and further supports the electrostatic interaction model of binding.
Thermal Gradient Gel Electrophoresis Analysis of Bioprotection from Pollutant Shocks in the Activated Sludge Microbial Community
Authors: Eichner Christine A. , Erb Rainer W. , Timmis Kenneth N. , Wagner-Döbler Irene
Published: December 2019

We used a culture-independent approach, namely, thermal gradient gel electrophoresis (TGGE) analysis of ribosomal sequences amplified directly from community DNA, to determine changes in the structure of the microbial community following phenol shocks in the highly complex activated sludge ecosystem. Parallel experimental model sewage plants were given shock loads of chlorinated and methylated phenols and simultaneously were inoculated (i) with a genetically engineered microorganism (GEM) able to degrade the added substituted phenols or (ii) with the nonengineered parental strain. The sludge community DNA was extracted, and 16S rDNA was amplified and analyzed by TGGE. To allow quantitative analysis of TGGE banding patterns, they were normalized to an external standard. The samples were then compared with each other for similarity by using the coefficient of Dice. The Shannon index of diversity,
H
, was calculated for each sludge sample, which made it possible to determine changes in community diversity. We observed a breakdown in community structure following shock loads of phenols by a decrease in the Shannon index of diversity from 1.13 to 0.22 in the noninoculated system. Inoculation with the GEM (
Pseudomonas
sp. strain B13 SN45RE) effectively protected the microbial community, as indicated by the maintenance of a high diversity throughout the shock load experiment (
H
decreased from 1.03 to only 0.82). Inoculation with the nonengineered parental strain,
Pseudomonas
sp. strain B13, did not protect the microbial community from being severely disturbed;
H
decreased from 1.22 to 0.46 for a 3-chlorophenol–4-methylphenol shock and from 1.03 to 0.70 for a 4-chlorophenol–4-methylphenol shock. The catabolic trait present in the GEM allowed for bioprotection of the activated sludge community from breakdown caused by toxic shock loading. In-depth TGGE analysis with similarity and diversity algorithms proved to be a very sensitive tool to monitor changes in the structure of the activated sludge microbial community, ranging from subtle shifts during adaptation to laboratory conditions to complete collapse following pollutant shocks.
Plasmid-Encoded Anthranilate Synthase (TrpEG) inBuchnera aphidicolafrom Aphids of the Family Pemphigidae
Authors: Van Ham Roeland C. H. J. , Martínez-Torres David , Moya Andrés , Latorre Amparo
Published: December 2019

Buchnera aphidicolais an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway inBuchnerafrom proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819–3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids inBuchnerafrom aphids of the distantly related family Pemphigidae.BuchnerafromTetraneura caerulescenscontained a 3.0-kb plasmid (pBTc2) that carried a single copy oftrpEGand resembledtrpEGplasmids ofBuchnerafrom the Aphididae. The second plasmid (pBPs2), isolated fromBuchneraofPemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designatedrepAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carryingrepAC1,trpG, and remnants oftrpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carryingrepAC2, the putative origin of replication, andtrpE. The unexpected finding of elements of the RepA/C replicon in previously characterizedtrpEGplasmids fromBuchneraof the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for allBuchnera trpEGamplifications.
A Hemorrhagic Factor (Apicidin) Produced by Toxic
Fusarium
Isolates from Soybean Seeds
Authors: Park Jun-Suk , Lee Kyung-Rim , Kim Jin-Cheol , Lim Sun-Hee , Seo Jeong-Ah , Lee Yin-Won
Published: December 2019

Fifty-two isolates of
Fusarium
species were obtained from soybean seeds from various parts of Korea and identified as
Fusarium oxysporum
,
F. moniliforme
,
F. semitectum
,
F. solani
,
F. graminearum
, or
F. lateritium
. These isolates were grown on autoclaved wheat grains and examined for toxicity in a rat-feeding test. Nine cultures were toxic to rats. One of these, a culture of
Fusarium
sp. strain KCTC 16677, produced apicidin, an antiprotozoal agent that caused toxic effects in rats (including body weight loss; hemorrhage in the stomach, intestines, and bladder; and finally death) when rats were fed diets supplemented with 0.05 and 0.1% apicidin. The toxin was toxic to brine shrimp (the 50% lethal concentration was 40 μg/ml) and was weakly cytotoxic to human and mouse tumor cell lines.
Glycerol Overproduction by EngineeredSaccharomyces cerevisiaeWine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase
Authors: Remize F. , Roustan J. L. , Sablayrolles J. M. , Barre P. , Dequin S.
Published: December 2019

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of theGPD1gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressingGPD1produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained byGPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.
Changes in Quinone Profiles of Hot Spring Microbial Mats with a Thermal Gradient
Authors: Hiraishi Akira , Umezawa Taichi , Yamamoto Hiroyuki , Kato Kenji , Maki Yonosuke
Published: December 2019

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68°C) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78°C hot spring sediment had a similar quinone profile.
Chloroflexus
-mixed mats occurred at temperatures of 61 to 65°C and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56°C were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50°C and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34°C were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (
D
), microbial divergence index (

MD
q

), and bioenergetic divergence index (

BD
q

). A
D
matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria,
Chloroflexus
spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of

MD
q

and

BD
q

values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.
In Situ Detection of Novel Bacterial Endosymbionts of
Acanthamoeba
spp. Phylogenetically Related to Members of the Order
Rickettsiales
Authors: Fritsche Thomas R. , Horn Matthias , Seyedirashti Seyedreza , Gautom Romesh K. , Schleifer Karl-Heinz , Wagner Michael
Published: December 2019

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the
Chlamydiales
. We report here the isolation from corneal scrapings of two
Acanthamoeba
strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the
Rickettsiales
branch of the alpha subdivision of the
Proteobacteria
. Nearest relatives included members of the genus
Rickettsia
, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the
Rickettsiales
. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most
Rickettsiales
with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.
Production of Wax Esters during Aerobic Growth of Marine Bacteria on Isoprenoid Compounds
Authors: Rontani Jean-Francois , Bonin Patricia C. , Volkman John K.
Published: December 2019

This paper describes the production of isoprenoid wax esters during the aerobic degradation of 6,10,14-trimethylpentadecan-2-one and phytol by four bacteria (
Acinetobacter
sp. strain PHY9,
Pseudomonas nautica
[IP85/617],
Marinobacter
sp. strain CAB [DSMZ 11874], and
Marinobacter hydrocarbonoclasticus
[ATCC 49840]) isolated from the marine environment. Different pathways are proposed to explain the formation of these compounds. In the case of 6,10,14-trimethylpentadecan-2-one, these esters result from the condensation of some acidic and alcoholic metabolites produced during the biodegradation, while phytol constitutes the alcohol moiety of most of the esters produced during growth on this isoprenoid alcohol. The amount of these esters formed increased considerably in N-limited cultures, in which the ammonium concentration corresponds to conditions often found in marine sediments. This suggests that the bacterial formation of isoprenoid wax esters might be favored in such environments. Although conflicting evidence exists regarding the stability of these esters in sediments, it seems likely that, under some conditions, bacterial esterification can enhance the preservation potential of labile compounds such as phytol.