bluuebunny/crossref_metadata_2025 huggingface dataset:
Ammonium Limitation Results in the Loss of Ammonia-Oxidizing Activity inNitrosomonas europaea
Authors: Stein Lisa Y. , Arp Daniel J.
Published: December 2019

The effects of limiting concentrations of ammonium on the metabolic activity ofNitrosomonas europaea, an obligate ammonia-oxidizing soil bacterium, were investigated. Cells were harvested during late logarithmic growth and were incubated for 24 h in growth medium containing 0, 15, or 50 mM ammonium. The changes in nitrite production and the rates of ammonia- and hydroxylamine-dependent oxygen consumption were monitored. In incubations without ammonium, there was little change in the ammonia oxidation activity after 24 h. With 15 mM ammonium, an amount that was completely consumed, there was an 85% loss of the ammonia oxidation activity after 24 h. In contrast, there was only a 35% loss of the ammonia oxidation activity after 24 h in the presence of 50 mM ammonium, an amount that was not consumed to completion. There was little effect on the hydroxylamine oxidation activity in any of the incubations. The loss of ammonia oxidation activity was not due to differences in steady-state levels of ammonia monooxygenase (AMO) mRNA (amoA) or to degradation of the active site-containing subunit of AMO protein. The incubations were also conducted at a range of pH values to determine whether the loss of ammonia oxidation activity was correlated to the residual ammonium concentration. The loss of ammonia oxidation activity after 24 h was less at lower pH values (where the unoxidized ammonium concentration was higher). When added in conjunction with limiting ammonium, short-chain alkanes, which are alternative substrates for AMO, prevented the loss of ammonia oxidation activity at levels corresponding to their binding affinity for AMO. These results suggest that substrates of AMO can preserve the ammonia-oxidizing activity ofN. europaeain batch incubations by protecting either AMO itself or other molecules associated with ammonia oxidation.
Effects of Cellular Metabolism and Viability on Metal Ion Accumulation by Cultured Biomass from a Bloom of the Cyanobacterium
Microcystis aeruginosa
Authors: Parker D. L. , Rai L. C. , Mallick N. , Rai P. K. , Kumar H. D.
Published: December 2019

The sorption of nickel, cadmium, and copper by cultured biomass from a naturally occurring bloom of
Microcystis aeruginosa
was demonstrated in two systems: cells suspended in culture medium and cells immobilized in alginate. Incubation in the absence of light, in the presence of metabolic inhibitors, and at 4°C did not substantially decrease the copper accumulation by cells in culture medium. Heat-killed, formaldehyde-treated, and air-dried biomass samples sorbed nearly as much (or in some cases slightly more) copper as did viable samples.
Changes in Permeability of Brush Border Membrane Vesicles from
Spodoptera littoralis
Midgut Induced by Insecticidal Crystal Proteins from
Bacillus thuringiensis
Authors: Escriche B. , De Decker N. , Van Rie J. , Jansens S. , Van Kerkhove E.
Published: December 2019

Bacillus thuringiensis
insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in
Spodoptera littoralis
, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.
Chemotaxis of Pathogenic
Vibrio
Strains towards Mucus Surfaces of Gilt-Head Sea Bream (
Sparus aurata
L.)
Authors: Bordas M. Angeles , Balebona M. Carmen , Rodriguez-Maroto Jose M. , Borrego Juan J. , Moriñigo Miguel A.
Published: December 2019

Vibrio anguillarum
and
Vibrio alginolyticus
exhibited significant adhesion to and chemotactic abilities towards mucus collected from the skin, gills, and intestine of gilt-head sea bream. Quadratic polynomial models for chemotaxis designed to estimate the influence of temperature demonstrated a differential bacterial chemotaxis depending of the source of the mucus, with the chemotaxis towards intestinal mucus being the least influenced.
High-Fidelity Translation of Recombinant Human Hemoglobin in
Escherichia coli
Authors: Weickert Michael J. , Apostol Izydor
Published: December 2019

Coexpression of di-α-globin and β-globin in
Escherichia coli
in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for
E. coli
proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ∼20% of the level of
E. coli
soluble proteins also resulted in equivalent translational fidelity.
Phylogeny and Identification In Situ of
Nevskia ramosa
Authors: Glöckner Frank Oliver , Babenzien Hans-Dietrich , Amann Rudolf
Published: December 2019

An enrichment of the neuston bacterium
Nevskia ramosa
was investigated by the cultivation-independent rRNA approach.
N. ramosa
was first described by Famintzin in 1892 as a rod-shaped, slightly bent bacterium forming typical flat rosettes on the surface of shallow freshwater habitats by unilateral slime formation. PCR in combination with cloning and sequencing was used for retrieving 21 partial and 5 nearly full-length 16S rRNA sequences forming three tight clusters. In situ hybridization with rRNA-targeted oligonucleotide probes allowed us to assign the three sequence clusters to three distinct bacterial populations abundant in the enrichment. The two probes that unambiguously identified the
N. ramosa
morphotype were derived from a 16S rRNA sequence that had similarities of 87.9 to 88.9% to the rRNA sequences of the most closely related group in the database,
Xanthomonas
sp. and relatives.
N. ramosa
currently is the only representative of an independent, deep branch of the gamma subclass of the class
Proteobacteria
. The two other populations abundant in the enrichment were affiliated with the alpha subclass of the class
Proteobacteria
. They were most closely related to
Blastobacter
sp. (97.2% similarity) and
Mycoplana bullata
(97.6% similarity) and might represent new species in the respective genera.
Phanerochaete chrysosporium
Cellobiohydrolase and Cellobiose Dehydrogenase Transcripts in Wood
Authors: Vallim Marcelo A. , Janse Bernard J. H. , Gaskell Jill , Pizzirani-Kleiner Aline A. , Cullen Daniel
Published: December 2019

The transcripts of structurally related cellobiohydrolase genes in
Phanerochaete chrysosporium
-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation.
Lysis of
Lactococcus lactis
subsp.
cremoris
SK110 and Its Nisin-Immune Transconjugant in Relation to Flavor Development in Cheese
Authors: Meijer Wilco , van de Bunt Bert , Twigt Marja , de Jonge Boudewijn , Smit Gerrit , Hugenholtz Jeroen
Published: December 2019

To develop a nisin-producing cheese starter,
Lactococcus lactis
subsp.
cremoris
SK110 was conjugated with transposon Tn
5276
-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn
5276
-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.
The Contribution of Caseins to the Amino Acid Supply for
Lactococcus lactis
Depends on the Type of Cell Envelope Proteinase
Authors: Flambard Benedicte , Helinck Sandra , Richard Jean , Juillard Vincent
Published: December 2019

The ability of caseins to fulfill the amino acid requirements of
Lactococcus lactis
for growth was studied as a function of the type of cell envelope proteinase (P
I
versus P
III
type). Two genetically engineered strains of
L. lactis
that differed only in the type of proteinase were grown in chemically defined media containing α
s1
-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for
L. lactis
in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the P
I
-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a P
III
-type proteinase.
Establishment of New Genetic Traits in a Microbial Biofilm Community
Authors: Christensen Bjarke B. , Sternberg Claus , Andersen Jens Bo , Eberl Leo , Møller Søren , Givskov Michael , Molin Søren
Published: December 2019

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species,
Pseudomonas putida
RI,
Acinetobacter
sp. strain C6, and an unidentified isolate, D8. Only
P. putida
RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated
lacI
q
gene and a
lacp-gfp
-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the
gfp
gene. This allowed a direct in situ identification of cells receiving the
gfp
-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of
P. putida
cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected
P. putida
RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting
P. putida
RI microcolonies in the biofilm.
Computation of the Electrical Double Layer Properties of Semipermeable Membranes in Multicomponent Electrolytes
Authors: Wasserman Evgeny , Felmy Andrew R.
Published: December 2019

A methodology is presented for calculating of the surface potential, Donnan potential, and ion concentration profiles for semipermeable microbial membranes that is valid for an arbitrary electrolyte composition. This model for surface potential, Donnan potential, and charge density was applied to recently reported experimental data for gram-positive bacteria, including
Bacillus brevis
,
Rhodococcus opacus
,
Rhodococcus erythropolis
, and
Corynebacterium
species. These calculations show that previously unconsidered trace amounts of divalent and trivalent cations at very low concentrations (10
−6
M) can have significant effects on the calculated surface and Donnan potentials, at ionic strengths of I ≤ 0.01 M, and that these effects need to be considered in accurate modeling of microbial surface. In addition, the calculated ion concentration profiles show that owing to the relatively high surface charges that can develop in microbial membranes, electrostatic effects can act to significantly concentrate divalent (factors of 5 × 10
3
) and trivalent (factors of 2 × 10
4
) cations within the bacterial cell wall. Comparison of the calculated concentration factors with those derived from experiments shows that a significant fraction of the uptake of metal by bacteria can be explained by the proposed electrostatic model.
Root Colonization by
Agrobacterium tumefaciens
Is Reduced in
cel
,
attB
,
attD
, and
attR
Mutants
Authors: Matthysse Ann G. , McMahan Susan
Published: December 2019

Root colonization by
Agrobacterium tumefaciens
was measured by using tomato and
Arabidopsis thaliana
roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 10
3
bacteria/cm of root length at the time of inoculation to more than 10
7
bacteria/cm after 10 days. The numbers of cellulose-minus and nonattaching
attB
,
attD
, and
attR
mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of
A. thaliana
ecotype Landsberg
erecta
, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of
A. tumefaciens
to cut
A. thaliana
roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with
cel
and
att
mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.
Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from
Leptospirillum ferrooxidans
Authors: Delgado Mónica , Toledo Héctor , Jerez Carlos A.
Published: December 2019

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium
Leptospirillum ferrooxidans
. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene
lcrI
for
Leptospirillum
chemotaxis receptor I. This is the first sequence reported for a gene from
L. ferrooxidans
encoding a protein. The
lcrI
gene showed both ς
28
-like and ς
70
-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from
Escherichia coli
), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in
E. coli
, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in
E. coli
. This was most likely due to the fact that the periplasmic pH of
E. coli
, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.
Cloning and Characterization of the Lactococcal Plasmid-Encoded Type II Restriction/Modification System,
Lla
DII
Authors: Madsen Annette , Josephsen Jytte
Published: December 2019

The
Lla
DII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in
Lactococcus lactis
subsp.
cremoris
W39. A 2.4-kb
Pst
I-
Eco
RI fragment inserted into the
Escherichia coli-L. lactis
shuttle vector pCI3340 conferred to
L. lactis
LM2301 and
L. lactis
SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The
Lla
DII endonuclease was partially purified and found to recognize and cleave the sequence 5′-GC↓NGC-3′, where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease
Fnu
4HI. Sequencing of the 2.4-kb
Pst
I-
Eco
RI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the
Lla
DII R/M system showed high homology to that of its only sequenced isoschizomer,
Bsp
6I from
Bacillus
sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the
Lla
DII and
Bsp
6I R/M systems are identical. Both methylases have two recognition sites (5′-GCGGC-3′ and 5′-GCCGC-3′) forming a putative stem-loop structure spanning part of the presumed −35 sequence and part of the intervening region between the −35 and −10 sequences. Alignment of the
Lla
DII and
Bsp
6I methylases with other m
5
C methylases showed that the protein primary structures possessed the same organization.
Modeling of Growth of
Lactobacillus sanfranciscensis
and
Candida milleri
in Response to Process Parameters of Sourdough Fermentation
Authors: Gänzle Michael G. , Ehmann Michaela , Hammes Walter P.
Published: December 2019

We investigated the effect of the ecological factors pH, temperature, ionic strength, and lactate, acetate, and ethanol levels on
Candida milleri
and two strains of
Lactobacillus sanfranciscensis
, organisms representative of the microflora of sourdough. A mathematical model describing the single and combined effects of these factors on the growth of these organisms was established in accordance with the following criteria: quality of fit, biological significance of the parameters, and applicability of the in vitro data to in situ processes. The growth rates of
L. sanfranciscensis
LTH1729 and LTH2581 were virtually identical under all conditions tested. These organisms tolerated >160 mmol of undissociated acetic acid per liter. Growth occurred in the pH range of 3.9 to 6.7 and was completely inhibited by 4% NaCl.
C. milleri
had a lower optimum temperature for growth (27°C) than the lactobacilli. The growth of the yeast was not affected by pH in the range of 3.5 to 7, and up to 8% NaCl was tolerated. Complete inhibition of growth occurred at 150 mmol of undissociated acetic acid per liter, but acetate at concentrations of up to 250 mmol/liter exerted virtually no effect. The model provides insight into factors contributing to the stability of the sourdough microflora and can facilitate the design of novel sourdough processes.
Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes
Authors: Rudi Knut , Skulberg Olav M. , Larsen Frank , Jakobsen Kjetill S.
Published: December 2019

A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genus
Microcystis
. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.
Enhanced Broth Media for Selective Growth of
Vibrio vulnificus
Authors: Hsu Wei-Yea , Wei Cheng-i , Tamplin Mark L.
Published: December 2019

Rapid detection of
Vibrio vulnificus
can be enhanced by optimizing the components of enrichment broth. PNC (5% peptone, 1% NaCl, and 0.08% cellobiose [pH 8.0]) enhanced the growth of
V. vulnificus
compared to alkaline peptone broth. PNCC (PNC with 1.0 to 4.1 U of colistin methanesulfonate per ml) increased the growth of low levels of
V. vulnificus
while suppressing non-target bacteria.
Design and Application of Two Rapid Screening Techniques for Isolation of Mn(IV) Reduction-Deficient Mutants of
Shewanella putrefaciens
Authors: Burnes Brian S. , Mulberry Michael J. , DiChristina Thomas J.
Published: December 2019

Chemical mutagenesis procedures and two newly developed rapid plate assays were used to identify two Mn(IV) reduction-deficient (Mnr) mutants of
Shewanella putrefaciens
. All eleven members of a set of previously isolated Fe(III) reduction-deficient (Fer) mutants displayed Mnr-positive phenotypes on the plate assays and were also capable of anaerobic growth on Mn(IV) as the sole terminal electron acceptor.
Microbial Characterization of Biological Filters Used for Drinking Water Treatment
Authors: Moll Deborah M. , Summers R. Scott , Breen Alec
Published: December 2019

The impact of preozonation and filter contact time (depth) on microbial communities was examined in drinking water biofilters treating Ohio River water which had undergone conventional treatment (coagulation, flocculation, sedimentation) or solutions of natural organic matter isolated from groundwater (both ozonated and nonozonated). With respect to filter depth, compared to filters treating nonozonated waters, preozonation of treated water led to greater differences in community phospholipid fatty acid (PLFA) profiles, utilization of sole carbon sources (Biolog), and arbitrarily primed PCR fingerprints. PLFA profiles indicated that there was a shift toward anaerobic bacteria in the communities found in the filter treating ozonated water compared to the communities found in the filter treating nonozonated settled water, which had a greater abundance of eukaryotic markers.
De Novo Synthesis of Amino Acids by the Ruminal Bacteria
Prevotella bryantii
B
1
4,
Selenomonas ruminantium
HD4, and
Streptococcus bovis
ES1
Authors: Atasoglu Cengiz , Valdés Carmen , Walker Nicola D. , Newbold C. James , Wallace R. John
Published: December 2019

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria,
Prevotella bryantii
B
1
4,
Selenomonas ruminantium
HD4, and
Streptococcus bovis
ES1, was determined by growing these bacteria in media containing
15
NH
4
Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by
P. bryantii
,
S. ruminantium
, and
S. bovis
, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of
P. bryantii
, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.
Aerobic Degradation of 2,4,6-Trinitrotoluene by
Enterobacter cloacae
PB2 and by Pentaerythritol Tetranitrate Reductase
Authors: French Christopher E. , Nicklin Stephen , Bruce Neil C.
Published: December 2019

Enterobacter cloacae
PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase.
E. cloacae
PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant
Escherichia coli
expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.
Deletion of the
rbo
Gene Increases the Oxygen Sensitivity of the Sulfate-Reducing Bacterium
Desulfovibrio vulgaris
Hildenborough
Authors: Voordouw Johanna K. , Voordouw Gerrit
Published: December 2019

The
rbo
gene of
Desulfovibrio vulgaris
Hildenborough encodes rubredoxin oxidoreductase (Rbo), a 14-kDa iron sulfur protein; forms an operon with the gene for rubredoxin; and is preceded by the gene for the oxygen-sensing protein DcrA. We have deleted the
rbo
gene from
D. vulgaris
with the
sacB
mutagenesis procedure developed previously (R. Fu and G. Voordouw, Microbiology 143:1815–1826, 1997). The absence of the
rbo
-gene in the resulting mutant,
D. vulgaris
L2, was confirmed by PCR and protein blotting with Rbo-specific polyclonal antibodies.
D. vulgaris
L2 grows like the wild type under anaerobic conditions. Exposure to air for 24 h caused a 100-fold drop in CFU of L2 relative to the wild type. The lag times of liquid cultures of inocula exposed to air were on average also greater for L2 than for the wild type. These results demonstrate that Rbo, which is not homologous with superoxide dismutase or catalase, acts as an oxygen defense protein in the anaerobic, sulfate-reducing bacterium
D. vulgaris
Hildenborough and likely also in other sulfate-reducing bacteria and anaerobic archaea in which it has been found.
Enzyme-Linked Immunofiltration Assay To Estimate Attachment of Thiobacilli to Pyrite
Authors: Dziurla Marie-Antoinette , Achouak Wafa , Lam Bach-Tuyet , Heulin Thierry , Berthelin Jacques
Published: December 2019

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically
Thiobacillus ferrooxidans
attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against
T. ferrooxidans
DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus
Thiobacillus
. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10
4
bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10
5
bacteria mg
−1
of pyrite at a particle size of 56 to 65 μm. The geometric coverage ratio of pyrite by
T. ferrooxidans
ranged from 0.25 to 2.25%. This suggests an attachment of
T. ferrooxidans
on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of
T. ferrooxidans
on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.
Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast
Authors: Wakisaka Shinji , Ohshima Yoshifumi , Ogawa Masahiro , Tochikura Tatsurokuro , Tachiki Takashi
Published: December 2019

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.
Genetic Variation in the Free-Living AmoebaNaegleria fowleri
Authors: Pélandakis Michel , De Jonckheere Johan F. , Pernin Pierre
Published: December 2019

In this study, 30 strains of the pathogenic free-living amoebaNaegleria fowleriwere investigated by using the randomly amplified polymorphic DNA (RAPD) method. The present study confirmed our previous finding that RAPD variation is not correlated with geographical origin. In particular, Mexican strains belong to the variant previously detected in Asia, Europe, and the United States. In France, surprisingly, strains from Cattenom gave RAPD patterns identical to those of the Japanese strains. In addition, all of these strains, together with an additional French strain from Chooz, exhibited similarities to South Pacific strains. The results also confirmed the presence of numerous variants in Europe, whereas only two variants were detected in the United States. The two variants found in the United States were different from the South Pacific variants. These findings do not support the previous hypothesis concerning the origin and modes of dispersal ofN. fowleri.
Conversion of Phenylalanine to Benzaldehyde Initiated by an Aminotransferase in
Lactobacillus plantarum
Authors: Nierop Groot Masja N. , de Bont Jan A. M.
Published: December 2019

The production of benzaldehyde from phenylalanine has been studied in various microorganisms, and several metabolic pathways have been proposed in the literature for the formation of this aromatic flavor compound. In this study, we describe benzaldehyde formation from phenylalanine by using a cell extract of
Lactobacillus plantarum
. Phenylalanine was initially converted to phenylpyruvic acid by an aminotransferase in the cell extract, and the keto acid was further transformed to benzaldehyde. However, control experiments with boiled cell extract revealed that the subsequent conversion of phenylpyruvic acid was a chemical oxidation step. It was observed that several cations could replace the extract in the conversion of phenylpyruvic acid to benzaldehyde. Addition of Cu(II) ions to phenylpyruvic acid resulted not only in the formation of benzaldehyde, but also in the generation of phenylacetic acid, mandelic acid, and phenylglyoxylic acid. These compounds have been considered intermediates in the biological conversion of phenylalanine. The chemical conversion step of phenylpyruvic acid was dependent on temperature, pH, the availability of cations, and the presence of oxygen.
Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge:
Nitrosococcus mobilis
and
Nitrospira
-Like Bacteria as Dominant Populations
Authors: Juretschko Stefan , Timmermann Gabriele , Schmid Markus , Schleifer Karl-Heinz , Pommerening-Röser Andreas , Koops Hans-Peter , Wagner Michael
Published: December 2019

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of
Nitrosococcus mobilis
-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of
N. mobilis
as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (
amoA
) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13
amoA
clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding
amoA
gene fragments of
Nitrosomonas europaea
Nm50 and the
N. mobilis
isolate. The unexpected high sequence similarity between the
amoA
gene fragments of the
N. mobilis
isolate and
N. europaea
indicates a possible lateral gene transfer event. Although a
Nitrobacter
strain was isolated, members of the nitrite-oxidizing genus
Nitrobacter
were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus
Nitrospira
. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that
Nitrospira
-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with
N. mobilis
.
Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization
Authors: Bidnenko Elena , Mercier Carine , Tremblay Josselyne , Tailliez Patrick , Kulakauskas Saulius
Published: December 2019

Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.
Screening of a Fosmid Library of Marine Environmental Genomic DNA Fragments Reveals Four Clones Related to Members of the Order
Planctomycetales
Authors: Vergin Kevin L. , Urbach Ena , Stein Jeffery L. , DeLong Edward F. , Lanoil Brian D. , Giovannoni Stephen J.
Published: December 2019

A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591–599, 1996) yielded four clones with 16S rRNA genes from the order
Planctomycetales
. Three of the clones belong to the
Pirellula
group and one clone belongs to the
Planctomyces
group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.
In Vitro and In Vivo Characterization of a Small-Colony Variant of the Primary Form of
Photorhabdus luminescens
MD (
Enterobacteriaceae
)
Authors: Hu Kaiji , Webster John M.
Published: December 2019

A small-colony variant (Vsm) of the primary form (Vp) of
Photorhabdus luminescens
MD from in vitro and in vivo cultures is described. Unlike the primary form, Vp, the Vsm variant is not the preferred diet of its nematode symbiont, a
Heterorhabditis
sp., does not support development and reproduction of the nematode, and is less pathogenic than Vp to
Galleria mellonella
larvae. Vsm cells were carried by 25% of infective juveniles, but they comprised a very low percentage (∼0.4%) of the total cells carried by the juvenile. In vitro subculture and in vivo injection into the larvae with either Vp or Vsm always produced a mixture of both Vp and Vsm. In nematode-bacterium-infected
G. mellonella
larvae, the Vp population in the hemocoel was high (4 × 10
9
to 5 × 10
9
CFU/g of wet insect tissue) at 24 h after infection, decreased about 10-fold by 48 h, and then regained a high level at day 5 before decreasing at day 7 and then remaining relatively constant through day 15 postinfection. The Vsm population, under the same conditions as those of Vp, increased gradually to a high level (9 × 10
8
CFU/g of wet insect tissue) at day 5 postinfection and then declined gradually through day 15.
Comparative Study of Pressure-Induced Germination of
Bacillus subtilis
Spores at Low and High Pressures
Authors: Wuytack Elke Y. , Boven Steven , Michiels Chris W.
Published: December 2019

We have studied pressure-induced germination of
Bacillus subtilis
spores at moderate (100 MPa) and high (500 to 600 MPa) pressures. Although we found comparable germination efficiencies under both conditions by using heat sensitivity as a criterion for germination, the sensitivity of pressure-germinated spores to some other agents was found to depend on the pressure used. Spores germinated at 100 MPa were more sensitive to pressure (>200 MPa), UV light, and hydrogen peroxide than were those germinated at 600 MPa. Since small, acid-soluble proteins (SASPs) and dipicolinic acid (DPA) are known to be involved in spore resistance to UV light and hydrogen peroxide, we studied the fate of these compounds during pressure germination. DPA was released upon both low- and high-pressure germination, but SASP degradation, which normally accompanies nutrient-induced germination, occurred upon low-pressure germination but not upon high-pressure germination. These results adequately explain the UV and hydrogen peroxide resistance of spores germinated at 600 MPa. The resistance to pressure inactivation of 600-MPa-germinated spores could also, at least partly, be attributed to α/β-type SASPs, since mutants deficient in α/β-type SASPs were more sensitive to inactivation at 600 MPa. Further, germination at 100 MPa resulted in rapid ATP generation, as is the case in nutrient-induced germination, but no ATP was formed during germination at 600 MPa. These results suggest that spore germination can be initiated by low- and high-pressure treatments but is arrested at an early stage in the latter case. The implications for the use of high pressure as a preservation treatment are discussed.
Confirmation of the Human-Pathogenic Microsporidia
Enterocytozoon bieneusi
,
Encephalitozoon intestinalis
, and
Vittaforma corneae
in Water
Authors: Dowd Scot E. , Gerba Charles P. , Pepper Ian L.
Published: December 2019

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular,
Enterocytozoon bieneusi
and
Encephalitozoon intestinalis
, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of
Encephalitozoon intestinalis
was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of
Enterocytozoon bieneusi
was confirmed in surface water; and the presence of
Vittaforma corneae
was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.
Assessment of Reductive Acetogenesis with Indigenous Ruminal Bacterium Populations and
Acetitomaculum ruminis
Authors: le Van Tricia D. , Robinson Joseph A. , Ralph John , Greening Richard C. , Smolenski Walter J. , Leedle Jane A. Z. , Schaefer Daniel M.
Published: December 2019

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H
2
disposal mechanism in the rumen. H
2
/CO
2
-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 × 10
5
cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH
13
CO
3
, and 100% H
2
gas phase since [U-
13
C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen
Acetitomaculum ruminis
190A4 (10
7
CFU/ml). To assess the relative importance of population density and/or H
2
concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N
2
gas phase. Both selective inhibition of methanogenesis and
A. ruminis
190A4 fortification (>10
5
CFU/ml) were necessary for the detection of reductive acetogenesis under H
2
-limiting conditions. Under these conditions, H
2
accumulated to 4,800 ppm. In contrast, H
2
accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H
2
concentrations correlated well with the pure culture H
2
threshold concentrations determined for
A. ruminis
190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H
2
partial pressure below the level necessary for H
2
utilization by
A. ruminis
190A4.
Sources of
Listeria monocytogenes
Contamination in a Cold-Smoked Rainbow Trout Processing Plant Detected by Pulsed-Field Gel Electrophoresis Typing
Authors: Autio Tiina , Hielm Sebastian , Miettinen Maria , Sjöberg Anna-Maija , Aarnisalo Kaarina , Björkroth Johanna , Mattila-Sandholm Tiina , Korkeala Hannu
Published: December 2019

Sites of
Listeria monocytogenes
contamination in a cold-smoked rainbow trout (
Oncorhynchus mykiss
) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing
L. monocytogenes
was low. During processing, the frequency of fish contaminated with
L. monocytogenes
clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating
L. monocytogenes
pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an
L. monocytogenes
eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating
L. monocytogenes
. None of the control samples taken in the 5 months after the eradication program was implemented contained
L. monocytogenes
.
Morphological and Biochemical Properties of a
Sphaerotilus
sp. Isolated From Paper Mill Slimes
Authors: Pellegrin Véronique , Juretschko Stefan , Wagner Michael , Cottenceau Gilles
Published: December 2019

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus
Sphaerotilus
. However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and
Sphaerotilus natans
DSM 6575
T
, DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of
S. natans
, all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of
S. natans
DSM 6575
T
and ATCC 15291. Polyphasic characterization of the isolated
Sphaerotilus
strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.