bluuebunny/crossref_metadata_2025 huggingface dataset:
A General Method for Selection of α-Acetolactate Decarboxylase-Deficient
Lactococcus lactis
Mutants To Improve Diacetyl Formation
Authors: Curic Mirjana , Stuer-Lauridsen Birgitte , Renault Pierre , Nilsson Dan
Published: December 2019

The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the
ilv
genes (encoding the enzymes involved in the biosynthesis of IV) of
L. lactis
NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable
L. lactis
strains.
Interactions between Nitrogen Fixation and Osmoregulation in the Methanogenic Archaeon
Methanosarcina barkeri
227
Authors: Brabban A. D. , Orcutt E. N. , Zinder S. H.
Published: December 2019

The nitrogenase enzyme complex of
Methanosarcina barkeri
227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for
Azotobacter vinelandii
and
Clostridium pasteurianum
nitrogenases. Na
+
and K
+
had equivalent effects, whereas Mg
2+
was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl
−
was more inhibitory than acetate was. Because
M. barkeri
227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N
2
than with NH
4
+
. Cells grown with N
2
and cells grown with NH
4
+
produced equal concentrations of α-glutamate at low salt concentrations and equal concentrations of
N
ɛ
-acetyl-β-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.
Tn
5
-Induced and Spontaneous Switching of
Sinorhizobium meliloti
to Faster-Swarming Behavior
Authors: Wei Xueming , Bauer Wolfgang D.
Published: December 2019

Tn
5
mutants of
Sinorhizobium meliloti
RMB7201 which swarmed 1.5 to 2.5 times faster than the parental strain in semisolid agar, moist sand, and viscous liquid were identified. These faster-swarming (FS) mutants outgrew the wild type 30- to 40-fold within 2 days in mixed swarm colonies. The FS mutants survived and grew as well as or better than the wild type under all of the circumstances tested, except in a soil matrix subjected to air drying. Exopolysaccharide (EPS) synthesis was reduced in each of the FS mutants when they were grown on defined succinate-nitrate medium, but the extent of reduction was different for each. It appears that FS behavior likely results from a modest, general derepression of motility involving an increased proportion of motile and flagellated cells and an increased average number of flagella per cell and increased average flagellar length. Spontaneous FS variants of RMB7201 were obtained at a frequency of about 1 per 10,000 to 20,000 cells by either enrichment from the periphery of swarm colonies or screening of colonies for reduced EPS synthesis on succinate-nitrate plates. The spontaneous FS variants and Tn
5
FS mutants were symbiotically effective and competitive in alfalfa nodulation. Reversion of FS variants to wild-type behavior was sporadic, indicating that reversion is affected by unidentified environmental factors. Based on phenotypic and molecular differences between individual FS variants and mutants, it appears that there may be multiple genetic configurations that result in FS behavior in RMB7201. The facile isolation of spontaneous FS variants of
Escherichia coli
and
Pseudomonas aeruginosa
indicates that switching to FS behavior may be fairly common among bacterial species. The substantial growth advantage of FS mutants and variants wherever nutrient gradients exist suggests that switching to FS forms may be an important behavioral adaptation in natural environments.
Pulsed-Light Inactivation of Food-Related Microorganisms
Authors: Rowan N. J. , MacGregor S. J. , Anderson J. G. , Fouracre R. A. , McIlvaney L. , Farish O.
Published: December 2019

The effects of high-intensity pulsed-light emissions of high or low UV content on the survival of predetermined populations of
Listeria monocytogenes
,
Escherichia coli
,
Salmonella enteritidis
,
Pseudomonas aeruginosa
,
Bacillus cereus
, and
Staphylococcus aureus
were investigated. Bacterial cultures were seeded separately on the surface of tryptone soya-yeast extract agar and were reduced by up to 2 or 6 log
10
orders with 200 light pulses (pulse duration, ∼100 ns) of low or high UV content, respectively (
P
< 0.001).
Isolation ofVibrio vulnificusSerovar E from Aquatic Habitats in Taiwan
Authors: Amaro Carmen , Hor Lien-I , Marco-Noales Ester , Bosque Teresa , Fouz Belén , Alcaide Elena
Published: December 2019

The existence of strains ofVibrio vulnificusserovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping afterHindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.
Electron Microscopy Studies of Cell-Wall-Anchored Cellulose (Avicel)-Binding Protein (AbpS) from
Streptomyces reticuli
Authors: Walter Stefan , Rohde Manfred , Machner Matthias , Schrempf Hildgund
Published: December 2019

Streptomyces reticuli
produces a 35-kDa cellulose (Avicel)-binding protein (AbpS) which interacts strongly with crystalline cellulose but not with soluble types of cellulose. Antibodies that were highly specific for the NH
2
-terminal part of AbpS were isolated by using truncated AbpS proteins that differed in the length of the NH
2
terminus. Using these antibodies for immunolabelling and investigations in which fluorescence, transmission electron, or immunofield scanning electron microscopy was used showed that the NH
2
terminus of AbpS protrudes from the murein layer of
S. reticuli
. Additionally, inspection of ultrathin sections of the cell wall, as well as biochemical experiments performed with isolated murein, revealed that AbpS is tightly and very likely covalently linked to the polyglucane layer. As AbpS has also been found to be associated with protoplasts, we predicted that a COOH-terminal stretch consisting of 17 hydrophobic amino acids anchors the protein to the membrane. Different amounts of AbpS homologues of several
Streptomyces
strains were synthesized.
The Nuclear Ribosomal DNA Intergenic Spacer as a Target Sequence To Study Intraspecific Diversity of the Ectomycorrhizal BasidiomyceteHebeloma cylindrosporumDirectly onPinusRoot Systems
Authors: Guidot Alice , Lumini Erica , Debaud Jean-Claude , Marmeisse Roland
Published: December 2019

Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomyceteHebeloma cylindrosporumwas studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individualPinus pinasterroot systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14P. pinasterstands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3motif. This internal polymorphic sequence could not be amplified from the genomes of nine otherHebelomaspecies. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individualH. cylindrosporuminfectedP. pinastermycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species.
Inhibition of
Vibrio anguillarum
by
Pseudomonas fluorescens
AH2, a Possible Probiotic Treatment of Fish
Authors: Gram Lone , Melchiorsen Jette , Spanggaard Bettina , Huber Ingrid , Nielsen Torben F.
Published: December 2019

To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain
Pseudomonas fluorescens
strain AH2 against the fish-pathogenic bacterium
Vibrio anguillarum
. As iron is important in virulence and bacterial interactions, the effect of
P. fluorescens
AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited
P. fluorescens
AH2 inhibited the growth of
V. anguillarum
, whereas sterile-filtered supernatants from iron-replete cultures of
P. fluorescens
AH2 did not.
P. fluorescens
AH2 inhibited the growth of
V. anguillarum
during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1,000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (
Oncorynchus mykiss
Walbaum) to
P. fluorescens
AH2 at a density of 10
5
CFU/ml for 5 days before a challenge with
V. anguillarum
at 10
4
to 10
5
CFU/ml for 1 h. Some fish were also exposed to
P. fluorescens
AH2 at 10
7
CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12°C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.
A Cold-Active Glucanase from the Ruminal Bacterium
Fibrobacter succinogenes
S85
Authors: Iyo Abiye H. , Forsberg Cecil W.
Published: December 2019

We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe
Fibrobacter succinogenes
S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25°C, and retains about 70% of its maximum activity at 0°C, while EGD has a temperature optimum of 35°C and retains only about 18% of its maximal activity at 0°C. When assayed at 4°C, CelG exhibits a 33-fold-higher k
cat
value and a 73-fold-higher physiological efficiency (k
cat
/

K
m

) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or that
F. succinogenes
originated from the marine environment.
Geographic Distribution and Genetic Diversity of
Ceanothus
-Infective
Frankia
Strains
Authors: Ritchie Nancy J. , Myrold David D.
Published: December 2019

Little is known about
Ceanothus
-infective
Frankia
strains because no
Frankia
strains that can reinfect the host plants have been isolated from
Ceonothus
spp. Therefore, we studied the diversity of the
Ceonothus
-infective
Frankia
strains by using molecular techniques.
Frankia
strains inhabiting root nodules of nine
Ceanothus
species were characterized. The
Ceanothus
species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of
Frankia
strains that infect
Ceanothus
spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3′ end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among the
Ceanothus
-infective
Frankia
strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the
Ceanothus
host species. Instead, the
Frankia
strains present were related to the sample collection locales.
Integrative Model for Binding of
Bacillus thuringiensis
Toxins in Susceptible and Resistant Larvae of the Diamondback Moth (
Plutella xylostella
)
Authors: Ballester Victoria , Granero Francisco , Tabashnik Bruce E. , Malvar Thomas , Ferré Juan
Published: December 2019

Insecticidal crystal proteins from
Bacillus thuringiensis
in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (
Plutella xylostella
) is the first insect to evolve resistance to
B. thuringiensis
in open-field populations. The only known mechanism of resistance to
B. thuringiensis
in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of
B. thuringiensis
toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of
B. thuringiensis
crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.
Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay
Authors: del Papa María F. , Balagué Laura J. , Sowinski Susana Castro , Wegener Caren , Segundo Eduardo , Abarca Francisco Martínez , Toro Nicolás , Niehaus Karsten , Pühler Alfred , Aguilar O. Mario , Martínez-Drets Gloria , Lagares Antonio
Published: December 2019

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported
Rhizobium
sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (
Medicago sativa
) inefficiently, the AT isolates also nodulated the common bean and
Leucaena leucocephala
, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N
2
-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.
Identification of
Cryptosporidium felis
in a Cow by Morphologic and Molecular Methods
Authors: Bornay-Llinares Fernando J. , da Silva Alexandre J. , Moura Iaci N. S. , Myjak Przemysław , Pietkiewicz Halina , Kruminis-Łozowska Wiesława , Graczyk Thaddeus K. , Pieniazek Norman J.
Published: December 2019

Apicomplexan
Cryptosporidium
parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as
Cryptosporidium baileyi
, which infects chickens, other species of this genus, such as
C. parvum
, infect a wide range of mammalian species from mice to humans. During an investigation of
Cryptosporidium
infection in cattle on a farm in northern Poland, we identified an infection caused by
C. felis
, in addition to known infections with
C. muris
and
C. parvum
. This new infection was identified based on the size of the oocysts (mean size, 4.3 ± 0.4 μm; range, 3.5 to 5.0 μm), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of
Cryptosporidium
species.
Cyclic-Imide-Hydrolyzing Activity of
d
-Hydantoinase from
Blastobacter
sp. Strain A17p-4
Authors: Soong Chee-Leong , Ogawa Jun , Honda Michinari , Shimizu Sakayu
Published: December 2019

The cyclic-imide-hydrolyzing activity of a prokaryotic cyclic-ureide-hydrolyzing enzyme,
d
-hydantoinase, was investigated. The enzyme hydrolyzed cyclic imides with bulky substituents such as 2-methylsuccinimide, 2-phenylsuccinimide, phthalimide, and 3,4-pyridine dicarboximide to the corresponding half-amides. However, simple cyclic imides without substituents, which are substrates of imidase (i.e., succinimide, glutarimide, and sulfur-containing cyclic imides such as 2,4-thiazolidinedione and rhodanine), were not hydrolyzed. The combined catalytic actions of bacterial
d
-hydantoinase and imidase can cover the function of a single mammalian enzyme, dihydropyrimidinase. Prokaryotic
d
-hydantoinase also catalyzed the dehydrative cyclization of the half-amide phthalamidic acid to the corresponding cyclic imide, phthalimide. The reversible hydrolysis of cyclic imides shown by prokaryotic
d
-hydantoinase suggested that, in addition to pyrimidine metabolism, it may also function in cyclic-imide metabolism.
Microbiology of the Oil Fly,
Helaeomyia petrolei
Authors: Kadavy Dana R. , Plantz Bradley , Shaw Christopher A. , Myatt Jill , Kokjohn Tyler A. , Nickerson Kenneth W.
Published: December 2019

Helaeomyia petrolei
larvae isolated from the asphalt seeps of Rancho La Brea in Los Angeles, Calif., were examined for microbial gut contents. Standard counts on Luria-Bertani, MacConkey, and blood agar plates indicated ca. 2 × 10
5
heterotrophic bacteria per larva. The culturable bacteria represented 15 to 20% of the total population as determined by acridine orange staining. The gut itself contained large amounts of the oil, had no observable ceca, and maintained a slightly acidic pH of 6.3 to 6.5. Despite the ingestion of large amounts of potentially toxic asphalt by the larvae, their guts sustained the growth of 100 to 1,000 times more bacteria than did free oil. All of the bacteria isolated were nonsporeformers and gram negative. Fourteen isolates were chosen based on representative colony morphologies and were identified by using the Enterotube II and API 20E systems and fatty acid analysis. Of the 14 isolates, 9 were identified as
Providencia rettgeri
and 3 were likely
Acinetobacter
isolates. No evidence was found that the isolates grew on or derived nutrients from the asphalt itself or that they played an essential role in insect development. Regardless, any bacteria found in the oil fly larval gut are likely to exhibit pronounced solvent tolerance and may be a future source of industrially useful, solvent-tolerant enzymes.
Disaccharides as a New Class of Nonaccumulated Osmoprotectants for
Sinorhizobium meliloti
Authors: Gouffi Kamila , Pica Nathalie , Pichereau Vianney , Blanco Carlos
Published: December 2019

Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for
Sinorhizobium meliloti
because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed
S. meliloti
cells. Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose. Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of
Rhizobium leguminosarum
biovars phaseoli and trifolii. Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism. All of the disaccharides that acted as powerful osmoprotectants in
S. meliloti
and
R. leguminosarum
also acted as very effective competitors of [
14
C]sucrose uptake in salt-stressed cultures of these bacteria. Conversely, disaccharides that were not osmoprotective for
S. meliloti
and
R. leguminosarum
did not inhibit sucrose uptake in these bacteria. Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria. Natural-abundance
13
C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed
S. meliloti
cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide
N
-acetylglutaminylglutamine amide. The ecological implication of the use of these disaccharides as osmoprotectants is discussed.
Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in
Enterococcus faecium
DPC1146
Authors: O’Keeffe Triona , Hill Colin , Ross R. Paul
Published: December 2019

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by
Enterococcus faecium
DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes
entA
,
entI
, and
entF
encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of
Escherichia coli
hemolysin A. Immediately downstream of the
entT
and
entD
genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the
yteI
gene of
Bacillus subtilis
and to
E. coli
protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the
entA
gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the
entF
gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in
Enterococcus faecalis
, while a four-gene construct (
entAITD
) under the control of a constitutive promoter resulted in heterologous enterocin A production in both
E. faecalis
and
Lactococcus lactis
.
Identification of Four Phage Resistance Plasmids from
Lactococcus lactis
subsp.
cremoris
HO2
Authors: Forde Amanda , Daly Charles , Fitzgerald Gerald F.
Published: December 2019

The bacteriophage-host sensitivity patterns of 16 strains of
Lactococcus lactis
originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates,
Lactococcus lactis
subsp.
cremoris
HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage φ712 (936 phage species) and the prolate-headed phage φc2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.
Degradation of Ciprofloxacin by Basidiomycetes and Identification of Metabolites Generated by the Brown Rot Fungus
Gloeophyllum striatum
Authors: Wetzstein Heinz-Georg , Stadler Marc , Tichy Hans-Volker , Dalhoff Axel , Karl Wolfgang
Published: December 2019

Ciprofloxacin (CIP), a fluoroquinolone antibacterial drug, is widely used in the treatment of serious infections in humans. Its degradation by basidiomycetous fungi was studied by monitoring
14
CO
2
production from [
14
C]CIP in liquid cultures. Sixteen species inhabiting wood, soil, humus, or animal dung produced up to 35%
14
CO
2
during 8 weeks of incubation. Despite some low rates of
14
CO
2
formation, all species tested had reduced the antibacterial activity of CIP in supernatants to between 0 and 33% after 13 weeks.
Gloeophyllum striatum
was used to identify the metabolites formed from CIP. After 8 weeks, mycelia had produced 17 and 10%
14
CO
2
from C-4 and the piperazinyl moiety, respectively, although more than half of CIP (applied at 10 ppm) had been transformed into metabolites already after 90 h. The structures of 11 metabolites were elucidated by high-performance liquid chromatography combined with electrospray ionization mass spectrometry and
1
H nuclear magnetic resonance spectroscopy. They fell into four categories as follows: (i) monohydroxylated congeners, (ii) dihydroxylated congeners, (iii) an isatin-type compound, proving elimination of C-2, and (iv) metabolites indicating both elimination and degradation of the piperazinyl moiety. A metabolic scheme previously described for enrofloxacin degradation could be confirmed and extended. A new type of metabolite, 6-defluoro-6-hydroxy-deethylene-CIP, provided confirmatory evidence for the proposed network of congeners. This may result from sequential hydroxylation of CIP and its congeners by hydroxyl radicals. Our findings reveal for the first time the widespread potential for CIP degradation among basidiomycetes inhabiting various environments, including agricultural soils and animal dung.
Variation in Resistance of Natural Isolates of
Escherichia coli
O157 to High Hydrostatic Pressure, Mild Heat, and Other Stresses
Authors: Benito Amparo , Ventoura Georgia , Casadei Maria , Robinson Tobin , Mackey Bernard
Published: December 2019

Strains of
Escherichia coli
O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of
E. coli
O157 when the efficacy of a novel or mild food preservation treatment is tested.
Selection of
clc
,
cba
, and
fcb
Chlorobenzoate-Catabolic Genotypes from Groundwater and Surface Waters Adjacent to the Hyde Park, Niagara Falls, Chemical Landfill
Authors: Peel Michelle C. , Wyndham R. Campbell
Published: December 2019

The frequency of isolation of three nonhomologous chlorobenzoate catabolic genotypes (
clc
,
cba
, and
fcb
) was determined for 464 isolates from freshwater sediments and groundwater in the vicinity of the Hyde Park industrial landfill site in the Niagara watershed. Samples were collected from both contaminated and noncontaminated sites during spring, summer, and fall and enriched at 4, 22, or 32°C with micromolar to millimolar concentrations of chlorobenzoates and 3-chlorobiphenyl (M. C. Peel and R. C. Wyndham, Microb. Ecol: 33:59–68, 1997). Hybridization at moderate stringency to restriction-digested genomic DNA with DNA probes revealed the chlorocatechol 1,2-dioxygenase operon (
clcABD
), the 3-chlorobenzoate 3,4-(4,5)-dioxygenase operon (
cbaABC
), and the 4-chlorobenzoate dehalogenase (
fcbB
) gene in isolates enriched from all contaminated sites in the vicinity of the industrial landfill. Nevertheless, the known genes were found in less than 10% of the isolates from the contaminated sites, indicating a high level of genetic diversity in the microbial community. The known genotypes were not enriched from the noncontaminated control sites nearby. The
clc
,
cba
, and
fcb
isolates were distributed across five phenotypically distinct groups based on Biolog carbon source utilization, with the breadth of the host range decreasing in the order
clc > cba > fcb
. Restriction fragment length polymorphism (RFLP) patterns showed that the
cba
genes were conserved in all isolates whereas the
clc
and
fcb
genes exhibited variation in RFLP patterns. These observations are consistent with the recent spread of the
cba
genes by horizontal transfer as part of transposon Tn
5271
in response to contaminant exposure at Hyde Park. Consistent with this hypothesis, IS
1071
, the flanking element in Tn
5271
, was found in all isolates that carried the
cba
genes. Interestingly, IS
1071
was also found in a high proportion of isolates from Hyde Park carrying the
clc
and
fcb
genes, as well as in type strains carrying the
clcABD
operon and the biphenyl (
bph
) catabolic genes.
Modes of Action of Acarbose Hydrolysis and Transglycosylation Catalyzed by a Thermostable Maltogenic Amylase, the Gene for Which Was Cloned from a
Thermus
Strain
Authors: Kim Tae-Jip , Kim Myo-Jeong , Kim Byung-Cheon , Kim Jae-Cherl , Cheong Tae-Kyou , Kim Jung-Wan , Park Kwan-Hwa
Published: December 2019

A maltogenic amylase gene was cloned in
Escherichia coli
from a gram-negative thermophilic bacterium,
Thermus
strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60°C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed β-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to
methyl
-α-
d
-glucopyranoside by forming an α-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.
Phenotypic Expression of PCR-Generated Random Mutations in a
Pseudomonas putida
Gene after Its Introduction into an
Acinetobacter
Chromosome by Natural Transformation
Authors: Kok Ruben G. , Young David M. , Ornston L. Nicholas
Published: December 2019

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of
Acinetobacter
ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to
Acinetobacter
genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an
Acinetobacter
chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of
Acinetobacter
DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered
Acinetobacter
recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich
Pseudomonas putida xylE
gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective
xylE
gene after growth at 37°C. Of recombinant clones that failed to express
xylE
at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.
Diversity of Nitrous Oxide Reductase (
nosZ
) Genes in Continental Shelf Sediments
Authors: Scala David J. , Kerkhof Lee J.
Published: December 2019

Diversity of the nitrous oxide reductase (
nosZ
) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the
nosZ
gene were amplified via PCR, using
nosZ
gene-specific primers. Thirty-seven unique copies of the
nosZ
gene from these marine environments were characterized, increasing the
nosZ
sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the
nosZ
gene was 64% ± 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (Cu
Z
) of the mature protein. Phylogenetic analysis demonstrated three major clusters of
nosZ
genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.
Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea Bacterium
Photobacterium profundum
SS9 at High Pressure and Low Temperature
Authors: Allen Eric E. , Facciotti Daniel , Bartlett Douglas H.
Published: December 2019

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium
Photobacterium profundum
SS9 were investigated. The fatty acids produced by
P. profundum
SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted.
P. profundum
SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5
n
-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.
Wide Distribution and Diversity of Members of the Bacterial Kingdom
Acidobacterium
in the Environment
Authors: Barns Susan M. , Takala Shannon L. , Kuske Cheryl R.
Published: December 2019

To assess the distribution and diversity of members of the recently identified bacterial kingdom
Acidobacterium
, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the
Acidobacterium
kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known
Proteobacteria
and gram-positive bacterial kingdoms.
Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ
Authors: Ouverney Cleber C. , Fuhrman Jed A.
Published: December 2019

We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines
3
H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class
Proteobacteria
(α-Proteobacteria) and of the
Cytophaga-Flavobacterium
group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and
Cytophaga-Flavobacterium
groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.
cumA
, a Gene Encoding a Multicopper Oxidase, Is Involved in Mn
2+
Oxidation in
Pseudomonas putida
GB-1
Authors: Brouwers Geert-Jan , de Vrind Johannes P. M. , Corstjens Paul L. A. M. , Cornelis Pierre , Baysse Christine , de Vrind-de Jong Elisabeth W.
Published: December 2019

Pseudomonas putida
GB-1-002 catalyzes the oxidation of Mn
2+
. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated
cumA
) encoding a protein homologous to multicopper oxidases. Addition of Cu
2+
increased the Mn
2+
-oxidizing activity of the
P. putida
wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu
2+
. A second open reading frame (designated
cumB
) is located downstream from
cumA
. Both
cumA
and
cumB
probably are part of a single operon. The translation product of
cumB
was homologous (level of identity, 45%) to that of
orf74
of
Bradyrhizobium japonicum
. A mutation in
orf74
resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the
cumA
mutant, suggesting that the
cumA
mutation may have a polar effect on
cumB
. This was confirmed by site-specific gene replacement in
cumB
. The
cumB
mutation did not affect the Mn
2+
-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn
2+
and that CumB is required for optimal growth of
P. putida
GB-1-002.