bluuebunny/crossref_metadata_2025_split huggingface dataset:
Construction and Expression of a Bifunctional Single-Chain Antibody againstBacillus cereusSpores
Authors: Koo Kai , Foegeding Peggy M. , Swaisgood Harold E. 
Published: December 2019

ABSTRACTThe variable-region genes of monoclonal antibody againstBacillus cereusspores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed inEscherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereusspore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.
Quantification of
Gordona amarae
Strains in Foaming Activated Sludge and Anaerobic Digester Systems with Oligonucleotide Hybridization Probes
Authors: de los Reyes M. Fiorella , de los Reyes Francis L. , Hernandez Mark , Raskin Lutgarde 
Published: December 2019

ABSTRACT

Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems.
Gordona
(formerly
Nocardia
)
amarae
often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four
G. amarae
strains were sequenced, and the resulting sequences were compared to the sequence of
G. amarae
type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for
G. amarae
, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional
G. amarae
isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of
G. amarae
, group 1, group 2, bacterial, mycolata, and
Gordona
rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain
G. amarae
strains may be regional or treatment plant specific and that previously uncharacterized
G. amarae
strains may be present in some systems.
Cloning of a
Sphingomonas paucimobilis
SYK-6 Gene Encoding a Novel Oxygenase That Cleaves Lignin-Related Biphenyl and Characterization of the Enzyme
Authors: Peng Xue , Egashira Takashi , Hanashiro Kaoru , Masai Eiji , Nishikawa Seiji , Katayama Yoshihiro , Kimbara Kazuhide , Fukuda Masao 
Published: December 2019

ABSTRACT

Sphingomonas paucimobilis
SYK-6 transforms 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb
Eco
RI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated
ligZ
. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the β subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704–2709, 1990), catechol 2,3-dioxygenase I (MpcI) of
Alcaligenes eutrophus
JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405–3406, 1990), the catalytic subunit of the
meta
-cleavage enzyme (CarBb) for 2′-aminobiphenyl-2,3-diol from
Pseudomonas
sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841–4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of
Escherichia coli
(E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249–5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting
meta
cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2′,3,3′-tetrahydroxy-5,5′-dicarboxybiphenyl and was dependent on the ferrous ion.
Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria
Authors: Stretton Serina , Techkarnjanaruk Somkiet , McLennan Alan M. , Goodman Amanda E. 
Published: December 2019

ABSTRACT

Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of
gfp
could be visualized in
Vibrio
sp. strain S141 cells at uniform levels of intensity from either the
lac
or the
npt
-2 promoter, whereas expression of
gfp
could be visualized in
Psychrobacter
sp. strain SW5H cells at various levels of intensity only from the
npt-
2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species,
Pseudoalteromonas
sp. strain S91, when the
gfp
gene was expressed from either promoter. A new mini-Tn
10
-
kan
-
gfp
transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn
10
-
kan
-
gfp
generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn
10
-
kan
-
gfp
) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn
10
-
kan
-
gfp
was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the
chi-gfp
fusion was induced in cells exposed to
N
′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of
chi
-negative, GFP
+
S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.
Intergeneric Transfer of Conjugative and Mobilizable Plasmids Harbored byEscherichia coliin the Gut of the Soil MicroarthropodFolsomia candida(Collembola)
Authors: Hoffmann Andrea , Thimm Torsten , Dröge Marcus , Moore Edward R. B. , Munch Jean Charles , Tebbe Christoph C. 
Published: December 2019

ABSTRACTThe gut of the soil microarthropodFolsomia candidaprovides a habitat for a high density of bacterial cells (T. Thimm, A. Hoffmann, H. Borkott, J. C. Munch, and C. C. Tebbe, Appl. Environ. Microbiol. 64:2660–2669, 1998). We investigated whether these gut bacteria act as recipients for plasmids fromEscherichia coli. Filter mating withE. colidonor cells and collected feces ofF. candidarevealed that the broad-host-range conjugative plasmid pRP4-luc (pRP4 with a luciferase marker gene) transferred to fecal bacteria at estimated frequencies of 5.4 × 10−1transconjugants per donor. The mobilizable plasmid pSUP104-luc was transferred from the IncQ mobilizing strainE. coliS17-1 and less efficiently from the IncF1 mobilizing strain NM522 but not from the nonmobilizing strain HB101. When S17-1 donor strains were fed toF. candida, transconjugants of pRP4-luc and pSUP104-luc were isolated from feces. Additionally, the narrow-host-range plasmid pSUP202-luc was transferred to indigenous bacteria, which, however, could not maintain this plasmid. Inhibition experiments with nalidixic acid indicated that pRP4-luc plasmid transfer took place in the gut rather than in the feces. A remarkable diversity of transconjugants was isolated in this study: from a total of 264 transconjugants, 15 strains belonging to the alpha, beta, or gamma subclass of the classProteobacteriawere identified by DNA sequencing of the PCR-amplified 16S rRNA genes and substrate utilization assays (Biolog). Except forAlcaligenes faecalis, which was identified by the Biolog assay, none of the isolates was identical to reference strains from data banks. This study indicates the importance of the microarthropod gut for enhanced conjugative gene transfer in soil microbial communities.
A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy
Authors: Mason David J. , Shanmuganathan Subo , Mortimer Fiona C. , Gant Vanya A. 
Published: December 2019

ABSTRACT
The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.
Algicidal Effects of a Novel Marine
Pseudoalteromonas
Isolate (Class
Proteobacteria
, Gamma Subdivision) on Harmful Algal Bloom Species of the Genera
Chattonella
,
Gymnodinium
, and
Heterosigma
Authors: Lovejoy Connie , Bowman John P. , Hallegraeff Gustaaf M. 
Published: December 2019

ABSTRACT

During a bacterial survey of the Huon Estuary in southern Tasmania, Australia, we isolated a yellow-pigmented
Pseudoalteromonas
strain (class
Proteobacteria
, gamma subdivision), designated strain Y, that had potent algicidal effects on harmful algal bloom species. This organism was identified by 16S rRNA sequencing as a strain with close affinities to
Pseudoalteromonas peptidysin
. This bacterium caused rapid cell lysis and death (within 3 h) of gymnodinoids (including
Gymnodinium catenatum
) and raphidophytes (
Chattonella marina
and
Heterosigma akashiwo
). It caused ecdysis of armored dinoflagellates (e.g.,
Alexandrium catenella
,
Alexandrium minutum
, and
Prorocentrum mexicanum
), but the algal cultures then recovered over the subsequent 24 h. Strain Y had no effect on a cryptomonad (
Chroomonas
sp.), a diatom (
Skeletonema
sp.), a cyanobacterium (
Oscillatoria
sp.), and two aplastidic protozoans. The algicidal principle of strain Y was excreted into the seawater medium and lost its efficacy after heating. Another common bacterial species,
Pseudoalteromonas carrageenovora
, was isolated at the same time and did not have these algicidal effects. The minimum concentrations of strain Y required to kill
G. catenatum
were higher than the mean concentrations found in nature under nonbloom conditions. However, the new bacterium showed a chemotactic, swarming behavior that resulted in localized high concentrations around target organisms. These observations imply that certain bacteria could play an important role in regulating the onset and development of harmful algal blooms.
Isolation of Broad-Host-Range Replicons from Marine Sediment Bacteria
Authors: Sobecky Patricia A. , Mincer Tracy J. , Chang Michelle C. , Toukdarian Aresa , Helinski Donald R. 
Published: December 2019

ABSTRACT

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888–895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the α and γ subclasses of the class
Proteobacteria
. Three of the marine sediment isolates are related to the γ-3 subclass organisms
Vibrio splendidus
and
Vibrio fischeri
, while the fourth isolate may be related to
Roseobacter litoralis
. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in
Escherichia coli
DH5α and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera
Achromobacter
,
Pseudomonas
,
Serratia
, and
Vibrio
. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.
Successive Mineralization and Detoxification of Benzo[
a
]pyrene by the White Rot Fungus
Bjerkandera
sp. Strain BOS55 and Indigenous Microflora
Authors: Kotterman Michiel J. J. , Vis Eric H. , Field Jim A. 
Published: December 2019

ABSTRACT

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH.
14
C-radiolabeled benzo[
a
]pyrene was subjected to oxidation by the white rot fungus
Bjerkandera
sp. strain BOS55. After 15 days, up to 8.5% of the [
14
C]benzo[
a
]pyrene was recovered as
14
CO
2
in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[
a
]pyrene on day 15 resulted in an initially rapid increase in the level of
14
CO
2
recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of
14
CO
2
recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in
14
CO
2
production were observed with several inocula, showing that some polar metabolites of fungal benzo[
a
]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[
a
]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[
a
]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the
Salmonella typhimurium
revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[
a
]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.
Inhibition of Anaerobic Phosphate Release by Nitric Oxide in Activated Sludge
Authors: Van Niel E. W. J. , Appeldoorn K. J. , Zehnder A. J. B. , Kortstee G. J. J. 
Published: December 2019

ABSTRACT

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P · g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-β-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating
Acinetobacter johnsonii
210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.
Differentiation of Dextran-Producing
Leuconostoc
Strains by a Modified Randomly Amplified Polymorphic DNA Protocol
Authors: Holt Scott M. , Cote Gregory L. 
Published: December 2019

ABSTRACT

Seven dextran-producing
Leuconostoc
strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes. RAPD profiles showed intraspecies differences among the
Leuconostoc mesenteroides
strains tested. This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.
Isolation, Characterization, and Heterologous Expression of the Novel Lantibiotic Epicidin 280 and Analysis of Its Biosynthetic Gene Cluster
Authors: Heidrich Christoph , Pag Ulrike , Josten Michaele , Metzger Jörg , Jack Ralph W. , Bierbaum Gabriele , Jung Günther , Sahl Hans-Georg 
Published: December 2019

ABSTRACT

Epicidin 280 is a novel type A lantibiotic produced by
Staphylococcus epidermidis
BN 280. During C
18
reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 ± 1.5 and 3,136 ± 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (
eciI
,
eciA
,
eciP
,
eciB
, and
eciC
, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of
Staphylococcus epidermidis
5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332–4338, 1994), was not detected. Instead, upstream of the immunity gene
eciI
we found an open reading frame,
eciO
, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host
Staphylococcus carnosus
TM 300 after introduction of
eciIAPBC
(i.e., no
eciO
was present) behaved homogeneously during reverse-phase chromatography.
Rapid Determination of Bacterial Abundance, Biovolume, Morphology, and Growth by Neural Network-Based Image Analysis
Authors: Blackburn Nicholas , Hagström Åke , Wikner Johan , Cuadros-Hansson Rocio , Bjørnsen Peter Koefoed 
Published: December 2019

ABSTRACT
Annual bacterial plankton dynamics at several depths and locations in the Baltic Sea were studied by image analysis. Individual bacteria were classified by using an artificial neural network which also effectively identified nonbacterial objects. Cell counts and frequencies of dividing cells were determined, and the data obtained agreed well with visual observations and previously published values. Cell volumes were measured accurately by comparison with bead standards. The survey included 690 images from a total of 138 samples. Each image contained approximately 200 bacteria. The images were analyzed automatically at a rate of 100 images per h. Bacterial abundance exhibited coherent patterns with time and depth, and there were distinct subsurface peaks in the summer months. Four distinct morphological classes were resolved by the image analyzer, and the dynamics of each could be visualized. The bacterial growth rates estimated from frequencies of dividing cells were different from the bacterial growth rates estimated by the thymidine incorporation method. With minor modifications, the image analysis technique described here can be used to analyze other planktonic classes.