bluuebunny/crossref_metadata_2025 huggingface dataset:
Antagonistic Activity of
Lactobacillus plantarum
C11: Two New Two-Peptide Bacteriocins, Plantaricins EF and JK, and the Induction Factor Plantaricin A
Authors: Anderssen Erlend L. , Diep Dzung Bao , Nes Ingolf F. , Eijsink Vincent G. H. , Nissen-Meyer Jon
Published: December 2019

Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by
Lactobacillus plantarum
C11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that
L. plantarum
C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 10
3
times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN.
Method Detection Limits of PCR and Immunofluorescence Assay forCryptosporidium parvumin Soil
Authors: Walker Mark J. , Montemagno Carlo , Bryant Juliet C. , Ghiorse William C.
Published: December 2019

We determined and compared the method detection limits (MDLα) of a PCR and an immunofluorescence assay (IFA) for detection ofCryptosporidium parvumoocysts in soils. Based on the MDLαand the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.
Detection of
Cyclospora cayetanensis
in Wastewater
Authors: Sturbaum Gregory D. , Ortega Ynes R. , Gilman Robert H. , Sterling Charles R. , Cabrera Lilia , Klein Donald A.
Published: December 2019

Cyclospora cayetanensis
causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate
Cyclospora
oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.
Bioencapsulation of Two DifferentVibrioSpecies in Nauplii of the Brine Shrimp (Artemia franciscana)
Authors: Gomez-Gil Bruno , Herrera-Vega Maria A. , Abreu-Grobois F. Alberto , Roque Ana
Published: December 2019

Two groups of nauplii from the brine shrimp (Artemia franciscana) were enriched with different bacteria, and the dynamics of bacterial uptake by the nauplii were observed. This study showed that the efficiency ofArtemia naupliiin bioencapsulating bacteria strongly depends on the type of bacteria used, time of exposure, and status (live or dead) of the bacteria.
Isolation and Entomotoxic Properties of the
Xenorhabdus nematophilus
F1 Lecithinase
Authors: Thaler Jacques-Olivier , Duvic Bernard , Givaudan Alain , Boemare Noël
Published: December 2019

Xenorhabdus
spp. and
Photorhabdus
spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that
Photorhabdus
spp. exhibited a broad lipase activity, while most of the
Xenorhabdus
spp. secreted a specific lecithinase.
Xenorhabdus
spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of
Xenorhabdus nematophilus
and
Xenorhabdus bovienii
strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a
X. nematophilus
F1 culture in two chromatographic steps, cation-exchange chromatography and C
18
reverse-phase chromatography. The substrate specificity of the
X. nematophilus
F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested,
Locusta migratoria
,
Galleria mellonella
,
Spodoptera littoralis
, and
Manduca sexta
. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.
Detoxification of Benzoxazolinone Allelochemicals from Wheat by
Gaeumannomyces graminis
var.
tritici
,
G. graminis
var.
graminis
,
G. graminis
var.
avenae
, and
Fusarium culmorum
Authors: Friebe A. , Vilich V. , Hennig L. , Kluge M. , Sicker D.
Published: December 2019

The ability of phytopathogenic fungi to overcome the chemical defense barriers of their host plants is of great importance for fungal pathogenicity. We studied the role of cyclic hydroxamic acids and their related benzoxazolinones in plant interactions with pathogenic fungi. We identified species-dependent differences in the abilities of
Gaeumannomyces graminis
var.
tritici
,
Gaeumannomyces graminis
var.
graminis
,
Gaeumannomyces graminis
var.
avenae
, and
Fusarium culmorum
to detoxify these allelochemicals of gramineous plants. The
G. graminis
var.
graminis
isolate degraded benzoxazolin-2(3
H
)-one (BOA) and 6-methoxy-benzoxazolin-2(3
H
)-one (MBOA) more efficiently than did
G. graminis
var.
tritici
and
G. graminis
var.
avenae. F. culmorum
degraded BOA but not MBOA.
N
-(2-Hydroxyphenyl)-malonamic acid and
N
-(2-hydroxy-4-methoxyphenyl)-malonamic acid were the primary
G. graminis
var.
graminis
and
G. graminis
var.
tritici
metabolites of BOA and MBOA, respectively, as well as of the related cyclic hydroxamic acids. 2-Amino-3
H
-phenoxazin-3-one was identified as an additional
G. graminis
var.
tritici
metabolite of BOA. No metabolite accumulation was detected for
G. graminis
var.
avenae
and
F. culmorum
by high-pressure liquid chromatography. The mycelial growth of the pathogenic fungi was inhibited more by BOA and MBOA than by their related fungal metabolites. The tolerance of
Gaeumannomyces
spp. for benzoxazolinone compounds is correlated with their detoxification ability. The ability of
Gaeumannomyces
isolates to cause root rot symptoms in wheat (cultivars Rektor and Astron) parallels their potential to degrade wheat allelochemicals to nontoxic compounds.
Role of Organic Acids in the Manganese-Independent Biobleaching System of
Bjerkandera
sp. Strain BOS55
Authors: Moreira María Teresa , Feijoo Gumersindo , Mester Tünde , Mayorga Pablo , Sierra-Alvarez Reyes , Field Jim A.
Published: December 2019

Bjerkandera
sp. strain BOS55 is a white rot fungus that can bleach EDTA-extracted eucalyptus oxygen-delignified kraft pulp (OKP) without any requirement for manganese. Under manganese-free conditions, additions of simple physiological organic acids (e.g., glycolate, glyoxylate, oxalate, and others) at 1 to 5 mM stimulated brightness gains and pulp delignification two- to threefold compared to results for control cultures not receiving acids. The role of the organic acids in improving the manganese-independent biobleaching was shown not to be due to pH-buffering effects. Instead, the stimulation was attributed to enhanced production of manganese peroxidase (MnP) and lignin peroxidase (LiP) as well as increased physiological concentrations of veratryl alcohol and oxalate. These factors contributed to greatly improved production of superoxide anion radicals, which may have accounted for the more extensive biobleaching. Optimum biobleaching corresponded most to the production of MnP. These results suggest that MnP from
Bjerkandera
is purposefully produced in the absence of manganese and can possibly function independently of manganese in OKP delignification. LiP probably also contributed to OKP delignification when it was present.
Construction and Expression of a Bifunctional Single-Chain Antibody againstBacillus cereusSpores
Authors: Koo Kai , Foegeding Peggy M. , Swaisgood Harold E.
Published: December 2019

The 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

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

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

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

The 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

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

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

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

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

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

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

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

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.
Biosensor Determination of the Microscale Distribution of Nitrate, Nitrate Assimilation, Nitrification, and Denitrification in a Diatom-Inhabited Freshwater Sediment
Authors: Lorenzen Jan , Larsen Lars Hauer , Kjær Thomas , Revsbech Niels-Peter
Published: December 2019

High-resolution NO
3
−
profiles in freshwater sediment covered with benthic diatoms were obtained with a new microscale NO
3
−
biosensor characterized by absence of interference from chemical species other than NO
2
−
and N
2
O. Analysis of the microprofiles obtained indicated no nitrification during darkness, high rates of nitrification and a tight coupling between nitrification and denitrification during illumination, and substantial rates of NO
3
−
assimilation during illumination. Nitrification during darkness could be induced by purging the bulk water with O
2
gas, indicating that the stimulatory effect on nitrification by illumination was caused by algal production of O
2
. NH
4
+
addition did not stimulate nitrification during darkness when O
2
was restricted to the upper 1-mm layer, and there was thus a low nitrification potential in the permanently oxic top 1 mm of the sediment.
Initial Transformations in the Biodegradation of Benzothiazoles by
Rhodococcus
Isolates
Authors: De Wever Helene , Vereecken Karen , Stolz Andreas , Verachtert Hubert
Published: December 2019

Benzothiazole-2-sulfonate (BTSO
3
) is one of the side products occurring in 2-mercaptobenzothiazole (MBT) production wastewater. We are the first to isolate an axenic culture capable of BTSO
3
degradation. The isolate was identified as a
Rhodococcus erythropolis
strain and also degraded 2-hydroxybenzothiazole (OBT) and benzothiazole (BT), but not MBT, which was found to inhibit the biodegradation of OBT, BT, and BTSO
3
. In anaerobic resting cell assays, BTSO
3
was transformed into OBT in stoichiometric amounts. Under aerobic conditions, OBT was observed as an intermediate in BT breakdown and an unknown compound transiently accumulated in several assays. This product was identified as a dihydroxybenzothiazole. Benzothiazole degradation pathways seem to converge into OBT, which is then transformed further into the dihydroxy derivative.
A New Intermediate in the Mineralization of 3,4-Dichloroaniline by the White Rot Fungus
Phanerochaete chrysosporium
Authors: Sandermann Heinrich , Heller Werner , Hertkorn Norbert , Hoque Enamul , Pieper Dietmar , Winkler Reinhard
Published: December 2019

Phanerochaete chrysosporium
ATCC 34541 has been reported to be unable to mineralize 3,4-dichloroaniline (DCA). However, high mineralization is now shown to occur when a fermentation temperature of 37° and gassing with oxygen are used. Mineralization did not correlate with lignin peroxidase activity. The latter was high under C limitation and low under N limitation, whereas the reverse was true for mineralization. The kinetics of DCA metabolism was studied in low-N and low-C and C- and N-rich culture media by metabolite analysis and
14
CO
2
determination. In all cases, DCA disappeared within 2 days, and a novel highly polar conjugate termed DCAX accumulated in the growth medium. This metabolite was a dead-end product under C and N enrichment. In oxygenated low-C medium and in much higher yield in oxygenated low-N medium, DCAX was converted to DCA-succinimide and then mineralized. DCAX was purified by high-performance liquid chromatography and identified as
N
-(3,4-dichlorophenyl)-α-ketoglutaryl-δ-amide by high-performance liquid chromatography and mass spectroscopy, gas chromatography and mass spectroscopy, and nuclear magnetic resonance spectroscopy. The formation of conjugate intermediates is proposed to facilitate mineralization because the sensitive amino group of DCA needs protection so that ring cleavage rather than oligomerization can occur.
Identification and Characterization of
Leuconostoc carnosum
, Associated with Production and Spoilage of Vacuum-Packaged, Sliced, Cooked Ham
Authors: Björkroth K. J. , Vandamme P. , Korkeala H. J.
Published: December 2019

Leuconostoc carnosum
was shown to be the specific spoilage organism in vacuum-packaged, sliced, cooked ham showing spoilage during 3 weeks of shelf life. Identification of the specific spoilage organism was done by use of phenotypic data and
Cla
I,
Eco
RI, and
Hin
dIII reference strain ribopatterns. One hundred
L. carnosum
isolates associated with the production and spoilage of the ham were further characterized by pulsed-field gel electrophoresis (PFGE), together with some meat-associated
Leuconostoc
species:
L. citreum
,
L. gelidum
,
L. mesenteroides
subsp.
dextranicum
, and
L. mesenteroides
subsp.
mesenteroides. Apa
I and
Sma
I digests divided the industrial
L. carnosum
strains into 25 different PFGE types,
Apa
I and
Sma
I types being consistent. Only one specific PFGE type was associated with the spoiled packages. This type also was detected in air and raw-meat mass samples. The spoilage strain did not produce bacteriocins. Only seven isolates belonging to three different PFGE types produced bacteriocins. Similarity analysis of the industrial
L. carnosum
strains revealed a homogeneous cluster which could be divided into eight subclusters consisting of strains having at most three-fragment differences. The
L. carnosum
cluster was clearly distinguished from the other meat-associated leuconostoc clusters, with the exception of the
L. carnosum
type strain. Ribotyping can be very helpful in the identification of
L. carnosum
, but its discriminatory power is too weak for strain characterization. PFGE provides good discrimination for studies dealing with the properties of homogeneous
L. carnosum
strains.
PCR-Based DNA Amplification and Presumptive Detection ofEscherichia coliO157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay
Authors: Oberst R. D. , Hays M. P. , Bohra L. K. , Phebus R. K. , Yamashiro C. T. , Paszko-Kolva C. , Flood S. J. A. , Sargeant J. M. , Gillespie J. R.
Published: December 2019

Presumptive identification ofEscherichia coliO157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagicE. coli(EHEC)eaeAgene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detectingE. coliO157:H7 DNA. The specificity of theeaeA-based 5′ nuclease assay system was sufficient to correctly identify allE. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection ofE. coliO157:H7 when ≥103CFU/ml was present in modified tryptic soy broth (mTSB) or modifiedE. colibroth and when ≥104CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification ofE. coliO157:H7 in ground beef and potentially in other food and environmental samples.
Genetic Relatedness among Environmental, Clinical, and Diseased-Eel
Vibrio vulnificus
Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR
Authors: Arias Covadonga R. , Pujalte María Jesús , Garay Esperanza , Aznar Rosa
Published: December 2019

Genetic relationships among 132 strains of
Vibrio vulnificus
(clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with
Kpn
I and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of
V. vulnificus
. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new
V. vulnificus
isolates.
Mode of Action of Linenscin OC2 against
Listeria innocua
Authors: Boucabeille Catherine , Letellier Lucienne , Simonet Jean-Marc , Henckes Gilles
Published: December 2019

Linenscin OC2 is a small hydrophobic substance produced by the orange cheese coryneform bacterium
Brevibacterium linens
OC2. Linenscin OC2 inhibits growth of gram-negative bacteria with an altered outer membrane permeability and gram-positive bacteria. It is also able to lyse eucaryotic cells. The mode of action of linenscin OC2 on the
Listeria innocua
cytoplasmic membrane and the effects of environmental parameters were investigated. Addition of low doses of linenscin OC2 resulted in an immediate perturbation of the permeability properties of the cytoplasmic membrane and of the bacterial energetic state. Linenscin OC2 induced a loss of cytoplasmic potassium, depolarization of the cytoplasmic membrane, complete hydrolysis of internal ATP, efflux of inorganic phosphate, and transient increase in oxygen consumption. Potassium loss occurred in the absence of a proton motive force and was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains of the cytoplasmic membrane. We propose that linenscin OC2 interacts with the cytoplasmic membrane and that the permeability changes observed at low doses reflect the formation of pore-like structures in this membrane.
Symbiotic Relationship of
Thiothrix
spp. with an Echinoderm
Authors: Brigmon Robin L. , De Ridder Chantal
Published: December 2019

Immunoassay procedures were used to investigate the symbiotic relationship of
Thiothrix
spp. in the intestinal cecum of the spatangoid species
Echinocardium cordatum. Thiothrix
spp. were identified in nodule samples from
E. cordatum
digestive tubes based on microscopic examination, enzyme-linked immunosorbent assay, and indirect immunofluorescence.
Thiothrix
spp. protein made up as much as 84% of the total protein content of the nodules. This is the first identification of
Thiothrix
spp. internally symbiotic with marine invertebrates.
Threonine Overproduction in Yeast Strains Carrying the
HOM3-R2
Mutant Allele under the Control of Different Inducible Promoters
Authors: Farfán María-José , Aparicio Luis , Calderón Isabel L.
Published: December 2019

The
HOM3
gene of
Saccharomyces cerevisiae
codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis. With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the
HOM3-R2
mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, P

GAL1

, P

CHA1

, P

CYC1-HSE2

, and P

GPH1

. The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions. Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed. Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation. Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors.
In Situ Population Dynamics of Bacterial Viruses in a Terrestrial Environment
Authors: Ashelford Kevin E. , Day Martin J. , Bailey Mark J. , Lilley Andrew K. , Fry John C.
Published: December 2019

Predation by bacteriophages is thought to control bacterial numbers and facilitate gene transfer among bacteria in the biosphere. A thorough understanding of phage population dynamics is therefore necessary if their significance in natural environments is to be fully appreciated. Here we describe the in situ population dynamics of three separate phage populations predating on separate bacterial species, living on the surface of field-grown sugar beet (Beta vulgarisvar. Amethyst), as recorded over a 9-month period. The distributions of the three phage populations were different and fluctuated temporally in 1996 (peak density, ∼103PFU g−1). One of these populations, predating on the indigenous phytosphere bacteriumSerratia liquefaciensCP6, consisted of six genetically distinct DNA phages that varied in relative abundance to the extent that an apparent temporal succession was observed between the two most abundant phages, ΦCP6-1 and ΦCP6-4.
Population Dynamics of Chesapeake Bay Virioplankton: Total-Community Analysis by Pulsed-Field Gel Electrophoresis
Authors: Wommack K. Eric , Ravel Jacques , Hill Russell T. , Chun Jongsik , Colwell Rita R.
Published: December 2019

Recognition of viruses as the most abundant component of aquatic microbial communities has stimulated investigations of the impact of viruses on bacterio- and phytoplankton host communities. From results of field studies to date, it is concluded that in most aquatic environments, a reduction in the number of bacteria on a daily basis is caused by viral infection. However, the modest amount of in situ virus-mediated mortality may be less significant than viral infection serving to maintain clonal diversity in the host communities directly, through gene transmission (i.e., transduction), and indirectly, by elimination of numerically dominant host species. If the latter mechanism for controlling community diversity prevails, then the overall structure of aquatic viral communities would be expected to change as well over short seasonal and spatial scales. To determine whether this occurs, pulsed-field gel electrophoresis (PFGE) was used to monitor the population dynamics of Chesapeake Bay virioplankton for an annual cycle (1 year). Virioplankton in water samples collected at six stations along a transect running the length of the bay were concentrated 100-fold by ultrafiltration. Viruses were further concentrated by ultracentrifugation, and the concentrated samples were embedded in agarose. PFGE analysis of virus DNA in the agarose plugs yielded several distinct bands, ranging from 50 to 300 kb. Principal-component and cluster analyses of the virus PFGE fingerprints indicated that changes in virioplankton community structure were correlated with time, geographical location, and extent of water column stratification. From the results of this study, it is concluded that, based on the dynamic nature of the Chesapeake Bay virioplankton community structure, the clonal diversity of bacterio- and phytoplankton host communities is an important component of the virus community.
Biosynthetic Pathway of Citrinin in the Filamentous Fungus
Monascus ruber
as Revealed by
13
C Nuclear Magnetic Resonance
Authors: Hajjaj Hassan , Klaébé Alain , Loret Marie O. , Goma Gérard , Blanc Philippe J. , François Jean
Published: December 2019

Carbon isotope distribution of [
13
C]citrinin from
Monascus ruber
incubated with [
13
C]acetate revealed that the biosynthesis of the toxin originated from a tetraketide, instead of a pentaketide as has been shown for
Penicillium
and
Aspergillus
species. The production of polyketide red pigments and citrinin by
M. ruber
may therefore be regulated at the level of the tetraketide branch point.