bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Nucleotide Sequencing of a
Staphylococcus aureus
Gene Encoding a Branched-Chain-Amino-Acid Transporter
Authors: Vijaranakul Uriwan , Xiong Anming , Lockwood Katherine , Jayaswal R. K.
Published: December 2019

We recently characterized a transposon-induced NaCl-sensitive mutant of
Staphylococcus aureus
(U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889–1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the
S. aureus
chromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated α-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first
brnQ
gene in staphylococci to be described.
Development and Testing of a Bacterial Biosensor for Toluene-Based Environmental Contaminants
Authors: Willardson Barry M. , Wilkins Jon F. , Rand Timothy A. , Schupp James M. , Hill Karen K. , Keim Paul , Jackson Paul J.
Published: December 2019

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator
xylR
from the TOL plasmid of
Pseudomonas putida
mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, P
u
. A reporter plasmid was constructed by placing the
luc
gene for firefly luciferase under the control of XylR and P
u
. When
Escherichia coli
cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited
K
1/2
values ranging from 39.0 ± 3.8 μM for 3-xylene to 2,690 ± 160 μM for 3-methylbenzylalcohol (means ± standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.
Utilization of Oligopeptides by
Listeria monocytogenes
Scott A
Authors: Verheul Annette , Rombouts Frank M. , Abee Tjakko
Published: December 2019

For effective utilization of peptides,
Listeria monocytogenes
possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 61:226–233, 1995). The present results reveal that
L. monocytogenes
possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from
L. monocytogenes
, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic
L. monocytogenes
might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since
Pseudomonas fragi
as well as
Bacillus cereus
was found to enhance the growth of
L. monocytogenes
to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from
Bacillus licheniformis
. The possible contribution of the oligopeptide transport system in the establishment of high numbers of
L. monocytogenes
cells in fermented milk products is discussed.
Survival of Infectious
Cryptosporidium parvum
Oocysts in Seawater and Eastern Oysters (
Crassostrea virginica
) in the Chesapeake Bay
Authors: Fayer Ronald , Graczyk Thaddeus K. , Lewis Earl J. , Trout James M. , Farley C. Austin
Published: December 2019

Oocysts of
Cryptosporidium parvum
placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters,
Crassostrea virginica
, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of
Cryptosporidium
oocysts and
Giardia
cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies.
Giardia
was not detected by this method from any of the 360 oysters examined. Presumptive identification of
Cryptosporidium
oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of
C. parvum
. These findings demonstrate for the first time that oysters in natural waters harbor infectious
C. parvum
oocysts and can serve as mechanical vectors of this pathogen.
Methane and Trichloroethylene Degradation by
Methylosinus trichosporium
OB3b Expressing Particulate Methane Monooxygenase
Authors: Lontoh Sonny , Semrau Jeremy D.
Published: December 2019

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on
Methylosinus trichosporium
OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For
M. trichosporium
OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (
V
max
) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (

K
s

) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane,
V
max
/

K
s

, doubled from 4.9 × 10
−3
to 9.9 × 10
−3
liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by
M. trichosporium
OB3b was also examined with varying copper and formate concentrations.
M. trichosporium
OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the
V
max
for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the
V
max
to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in

K
s

from 36 to 7.9 μM.
V
max
/

K
s

for TCE degradation by pMMO also increased from 6.9 × 10
−5
to 5.2 × 10
−4
liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.
Potential of Unicellular Cyanobacteria from Saline Environments as Exopolysaccharide Producers
Authors: De Philippis Roberto , Margheri Maria Cristina , Materassi Riccardo , Vincenzini Massimo
Published: December 2019

Fifteen
Cyanothece
strains isolated from saline environments have been characterized with regard to exopolysaccharide (EPS) production. The polymers contained six to eight monosaccharides, with one or two acidic sugars. In some EPS samples, the additional presence of acetyl, pyruvyl, and/or sulfate groups was also detected.
Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase
Authors: Feller Georges , Le Bussy Olivier , Gerday Charles
Published: December 2019

α-Amylase from the antarctic psychrophile
Alteromonas haloplanktis
is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in
Escherichia coli
, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and
E. coli
growth rate is reached at 18°C.
Light-Mediated Nitrite Accumulation during Denitrification by
Pseudomonas
sp. Strain JR12
Authors: Barak Yoram , Tal Yossi , van Rijn Jaap
Published: December 2019

The effect of light on the denitrifying characteristics of a nonphotosynthetic denitrifier,
Pseudomonas
sp. strain JR12, was examined. Already at low light intensities, nitrite accumulated as a result of light inhibition of nitrite but not of nitrate reduction rates. Exposure of this bacterium to light caused a photooxidation of cytochrome
c
, an intermediate electron carrier in its respiratory pathway. Photoinhibition of nitrite reduction was reversible, as nitrite reduction rates returned to preillumination levels when light-exposed cells were returned to dark conditions. Antimycin A reversed the inhibitory effect of light on nitrite reduction by preventing a reversed electron flow. Aerobic respiration by this bacterium was not affected by light.
Cloning and Sequencing of the
Sphingomonas
(
Pseudomonas
)
paucimobilis
Gene Essential for the O Demethylation of Vanillate and Syringate
Authors: Nishikawa Seiji , Sonoki Tomonori , Kasahara Tatsuhide , Obi Takahiro , Kubota Shoko , Kawai Shinya , Morohoshi Noriyuki , Katayama Yoshihiro
Published: December 2019

Sphingomonas
(
Pseudomonas
)
paucimobilis
SYK-6 is able to grow on 5,5′-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of
S. paucimobilis
SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2′,3′-trihydroxy-3-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-
O
-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of
Clostridium thermoaceticum.
Transcription of
ppk
from
Acinetobacter
sp. Strain ADP1, Encoding a Putative Polyphosphate Kinase, Is Induced by Phosphate Starvation
Authors: Geißdörfer Walter , Ratajczak Andreas , Hillen Wolfgang
Published: December 2019

Polyphosphate kinase (Ppk) catalyzes the formation of polyphosphate from ATP. We cloned the
ppk
gene (2,073 bp) from
Acinetobacter
sp. strain ADP1; this gene encodes a putative polypeptide of 78.6 kDa with extensive homology to polyphosphate kinase from
Escherichia coli
and other bacteria. Chromosomal disruption of
ppk
by inserting a transcriptionally fused
lacZ
does not affect growth under conditions of phosphate limitation or excess. β-Galactosidase activity expressed from the single-copy
ppk
::
lacZ
fusion is induced 5- to 15-fold by phosphate starvation. An increased amount of
ppk
transcript (2.2 kb) was detected when cells were grown at a limiting phosphate concentration. Primer extension analysis revealed a regulated promoter located upstream of a second, constitutive promoter. Potential similarities of this regulation with the effects of PhoB and PhoR of
E. coli
are discussed.
Biochemical and Genetic Characterization of an Extracellular Protease from
Pseudomonas fluorescens
CY091
Authors: Liao Ching-Hsing , McCallus Daniel E.
Published: December 2019

Pseudomonas fluorescens
CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl
2
or SrCl
2
but not in media containing ZnCl
2
, MgCl
2
, or MnCl
2
. The requirement of Ca
2+
(or Sr
2+
) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of
P. fluorescens
CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the
aprX
gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in
P. fluorescens
and
Escherichia coli
. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by
Pseudomonas aeruginosa
and
Erwinia chrysanthemi
. Two conserved sequence domains possibly associated with Ca
2+
and Zn
2+
binding were identified. Immediately adjacent to the
aprX
structural gene, a gene (
inh
) encoding a putative protease inhibitor and three genes (
aprD
,
aprE
, and
aprF
), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in
P. fluorescens
CY091 appears to be somewhat different from that previously demonstrated in
P. aeruginosa
and
E. chrysanthemi.
Acidophilic Methanotrophic Communities from
Sphagnum
Peat Bogs
Authors: Dedysh Svetlana N. , Panikov Nicolai S. , Tiedje James M.
Published: December 2019

Highly enriched methanotrophic communities (>25 serial transfers) were obtained from acidic ombrotrophic peat bogs from four boreal forest sites. The enrichment strategy involved using media conditions that were associated with the highest rates of methane uptake by the original peat samples, namely, the use of diluted mineral medium of low buffering capacity, moderate incubation temperature (20°C), and pH values of 3 to 6. Enriched communities contained a mixture of rod-shaped bacteria arranged in aggregates with a minor contribution of
Hyphomicrobium
-like cells. The growth stoichiometry of isolates was characteristic of methanotrophic bacteria (CH
4
/O
2
/CO
2
=1:1.1:0.59), with an average apparent yield of 0.41 ± 0.03 g of biomass C/g of CH
4
-C. DNA from each enrichment yielded a PCR product of the expected size with primers for both
mmoX
and
mmoY
genes of soluble methane monooxygenase. Two types of sequences were obtained for PCR-amplified fragments of
mmoX
. One of them exhibited high identity to the
mmoX
protein of the
Methylocystis-Methylosinus
group, whereas the other showed an equal level of divergence from both the
Methylosinus-Methylocystis
group and
Methylococcus capsulatus
(Bath) and formed a distinct branch. The pH optimum for growth and for CH
4
uptake was 4.5 to 5.5, which is very similar to that for the optimum CH
4
uptake observed in the original peat samples. These methanotrophs are moderate acidophiles rather than acidotolerant organisms, since their growth rate and methane uptake were much lower at neutral pH. The growth of the methanotrophic community was enhanced by using media with a very low salt content (20 to 200 mg/liter), more typical of their natural environment. All four enriched communities grew on N-free medium.
Bacterial Community Dynamics during Start-Up of a Trickle-Bed Bioreactor Degrading Aromatic Compounds
Authors: Stoffels Marion , Amann Rudolf , Ludwig Wolfgang , Hekmat Dariusch , Schleifer Karl-Heinz
Published: December 2019

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a car painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to
Pseudomonas putida
or
Pseudomonas mendocina
, which both belong to the gamma subclass of the class
Proteobacteria
. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera
Burkholderia
and
Sutterella
, probe ALBO34a for the genera
Alcaligenes
and
Bordetella
, and probe Bcv13b for
Burkholderia cepacia
and
Burkholderia vietnamiensis
. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.
Self-Transmissible Mercury Resistance Plasmids with Gene-Mobilizing Capacity in Soil Bacterial Populations: Influence of Wheat Roots and Mercury Addition
Authors: Smit Eric , Wolters Anneke , van Elsas Jan Dirk
Published: December 2019

A set of mercury resistance plasmids was obtained from wheat rhizosphere soil amended or not amended with mercuric chloride via exogenous plasmid isolation by using
Pseudomonas fluorescens
R2f,
Pseudomonas putida
UWC1, and
Enterobacter cloacae
BE1 as recipient strains. The isolation frequencies were highest from soil amended with high levels of mercury, and the isolation frequencies from unamended soil were low. With
P. putida
UWC1 as the recipient, the isolation frequency was significantly enhanced in wheat rhizosphere compared to bulk soil. Twenty transconjugants were analyzed per recipient strain. All of the transconjugants contained plasmids which were between 40 and 50 kb long. Eight selected plasmids were distributed among five groups, as shown by restriction digestion coupled with a similarity matrix analysis. However, all of the plasmids formed a tight group, as judged by hybridization with two whole-plasmid probes and comparisons with other plasmids in dot blot hybridization analyses. The results of replicon typing and broad-host-range incompatibility (Inc) group-specific PCR suggested that the plasmid isolates were not related to any previously described Inc group. Although resistance to copper, resistance to streptomycin, and/or resistance to chloramphenicol was found in several plasmids, catabolic sequences were generally not identified. One plasmid, pEC10, transferred into a variety of bacteria belonging to the β and γ subdivisions of the class
Proteobacteria
and mobilized as well as retromobilized the IncQ plasmid pSUP104. A PCR method for detection of pEC10-like replicons was used, in conjunction with other methods, to monitor pEC10-homologous sequences in mercury-polluted and unpolluted soils. The presence of mercury enhanced the prevalence of pEC10-like replicons in soil and rhizosphere bacterial populations.
PCR for Detection of
Shigella
spp. in Mayonnaise
Authors: Villalobo Eduardo , Torres Antonio
Published: December 2019

The use of PCR to amplify a specific
virA
gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus
Shigella
and enteroinvasive
Escherichia coli
. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of
Shigella
, individual cells of
Shigella dysenteriae
, and mayonnaise contaminated with
S. dysenteriae
. Moreover, a multiplex PCR with specific (
virA
) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for
Shigella
and enteroinvasive
E. coli.
Grazing of a
Tetrahymena
sp. on Adhered Bacteria in Percolated Columns Monitored by In Situ Hybridization with Fluorescent Oligonucleotide Probes
Authors: Eisenmann Heinrich , Harms Hauke , Meckenstock Rainer , Meyer Elisabeth I. , Zehnder Alexander J. B.
Published: December 2019

Predation of attached
Pseudomonas putida
mt2 by the small ciliate
Tetrahymena
sp. was investigated with a percolated column system. Grazing rates were examined under static and dynamic conditions and were compared to grazing rates in batch systems containing suspended prey. The prey densities were 2 × 10
8
bacteria per ml of pore space and 2 × 10
8
bacteria per ml of suspension, respectively. Postingestion in situ hybridization of bacteria with fluorescent oligonucleotide probes was used to quantify ingestion. During 30 min, a grazing rate of 1,382 ± 1,029 bacteria individual
−1
h
−1
was obtained with suspended prey; this was twice the grazing rate observed with attached bacteria under static conditions. Continuous percolation at a flow rate of 73 cm h
−1
further decreased the grazing rate to about 25% of the grazing rate observed with suspended prey. A considerable proportion of the protozoans fed on neither suspended bacteria nor attached bacteria. The transport of ciliates through the columns was monitored at the same time that predation was monitored. Less than 20% of the protozoans passed through the columns without being retained. Most of these organisms ingested no bacteria, whereas the retained protozoans grazed more efficiently. Retardation of ciliate transport was greater in columns containing attached bacteria than in bacterium-free columns. We propose that the correlation between grazing activity and retardation of transport is a consequence of the interaction between active predators and attached bacteria.
Characterization of the
rrnB
Operon of the Plant Pathogen
Rhodococcus fascians
and Targeted Integrations of Exogenous Genes at
rrn
Loci
Authors: Pisabarro Agustín , Correia António , Martín Juan F.
Published: December 2019

A 6.0-kb
Sal
I DNA fragment containing an entire rRNA operon (
rrnB
) was cloned from a cosmid gene bank of the phytopathogenic strain
Rhodococcus fascians
D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5′ and 3′ ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the
rrn
operons of
Bacillus subtilis
and other gram-positive bacteria. Four copies of the
rrn
operons were identified by hybridization with an
rrnB
probe in
R. fascians
type strain ATCC 12974 and in the virulent strain
R. fascians
D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified as
R. fascians
, produced five
rrn
-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to
rrnB
was constructed for targeted integration of exogenous genes at the
rrn
loci. Transformants carrying the exogenous chloramphenicol resistance gene (
cmr
) integrated in different
rrn
operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.
Effects of Ionic and Osmotic Strength on the Glucosyltransferase of
Rhizobium meliloti
Responsible for Cyclic β-(1,2)-Glucan Biosynthesis
Authors: Ingram-Smith Cheryl , Miller Karen J.
Published: December 2019

The cyclic β-(1,2)-glucans of
Rhizobium meliloti
and
Agrobacterium tumefaciens
play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic β-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from
R. meliloti
. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an
ndvA
mutant defective for transport of cyclic glucans and an
ndvB
mutant that produces a C-terminal truncated glucosyltransferase. Cyclic β-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic β-(1,2)-glucan biosynthesis.
Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by
Leuconostoc mesenteroides
NRRL B-1299
Authors: Dols Marguerite , Remaud-Simeon M. , Willemot R. M. , Vignon M. , Monsan P.
Published: December 2019

When grown in glucose or fructose medium in the absence of sucrose,
Leuconostoc mesenteroides
NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.
Seasonal Pattern of Tomato Mosaic Tobamovirus Infection and Concentration in Red Spruce Seedlings
Authors: Bachand George D. , Castello John D.
Published: December 2019

Tomato mosaic tobamovirus (ToMV) infects red spruce (
Picea rubens
) and causes significant changes in its growth and physiology. The mechanism of infection and the pattern of virus concentration in seedling roots and needles were investigated. One-year-old red spruce seedlings were obtained from the nursery in April and June 1995 and August 1996 and tested for ToMV using enzyme-linked immunosorbent assay (ELISA). Virus-free seedlings were divided into three treatments: control, root inoculated, and needle inoculated. Two control, five root-inoculated, and five needle-inoculated seedlings were sampled destructively at biweekly intervals for 3 months and then tested for ToMV by ELISA. ToMV was transmitted to seedlings by root but not by needle inoculation. The virus was detected in 67 to 100% of roots but in less than 7% of needles of root-inoculated seedlings. The percent infection of root-inoculated seedlings differed significantly between the April and June and between the April and August inoculation periods. Virus concentration in infected seedling roots increased initially, peaked within 4 weeks postinoculation, and steadily declined thereafter. Significant differences in ToMV concentrations in roots also were detected among inoculation periods and sampling dates. Early spring may represent the optimal time for infection of seedlings, as well as for assaying roots for ToMV.
Influence of Water Temperature and Salinity on
Vibrio vulnificus
in Northern Gulf and Atlantic Coast Oysters (
Crassostrea virginica
)
Authors: Motes M. L. , DePaola A. , Cook D. W. , Veazey J. E. , Hunsucker J. C. , Garthright W. E. , Blodgett R. J. , Chirtel S. J.
Published: December 2019

This study investigated the temperature and salinity parameters associated with waters and oysters linked to food-borne
Vibrio vulnificus
infections.
V. vulnificus
was enumerated in oysters collected at three northern Gulf Coast sites and two Atlantic Coast sites from July 1994 through September 1995. Two of these sites, Black Bay, La., and Apalachicola Bay, Fla., are the source of the majority of the oysters implicated in
V. vulnificus
cases. Oysters in all Gulf Coast sites exhibited a similar seasonal distribution of
V. vulnificus
: a consistently large number (median concentration, 2,300 organisms [most probable number] per g of oyster meat) from May through October followed by a gradual reduction during November and December to ≤10 per g, where it remained from January through mid-March, and a sharp increase in late March and April to summer levels.
V. vulnificus
was undetectable (<3 per g) in oysters from the North and South Carolina sites for most of the year. An exception occurred when a late-summer flood caused a drop in salinity in the North Carolina estuary, apparently causing
V. vulnificus
numbers to increase briefly to Gulf Coast levels. At Gulf Coast sites,
V. vulnificus
numbers increased with water temperatures up to 26°C and were constant at higher temperatures. High
V. vulnificus
levels (>10
3
per g) were typically found in oysters from intermediate salinities (5 to 25 ppt). Smaller
V. vulnificus
numbers (<10
2
per g) were found at salinities above 28 ppt, typical of Atlantic Coast sites. On 11 occasions oysters were sampled at times and locations near the source of oysters implicated in 13
V. vulnificus
cases; the
V. vulnificus
levels and environmental parameters associated with these samples were consistent with those of other study samples collected from the Gulf Coast from April through November. These findings suggest that the hazard of
V. vulnificus
infection is not limited to brief periods of unusual abundance of
V. vulnificus
in Gulf Coast oysters or to environmental conditions that are unusual to Gulf Coast estuaries.
Gene Cloning, Transcriptional Analysis, Purification, and Characterization of Phenolic Acid Decarboxylase from
Bacillus subtilis
Authors: Cavin Jean-François , Dartois Véronique , Diviès Charles
Published: December 2019

Bacillus subtilis
displays a substrate-inducible decarboxylating activity with the following three phenolic acids: ferulic,
p
-coumaric, and caffeic acids. Based on DNA sequence homologies between the
Bacillus pumilus
ferulate decarboxylase gene (
fdc
) (A. Zago, G. Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484–4486, 1995) and the
Lactobacillus plantarum p
-coumarate decarboxylase gene (
pdc
) (J.-F. Cavin, L. Barthelmebs, and C. Diviès, Appl. Environ. Microbiol. 63:1939–1944, 1997), a DNA probe of about 300 nucleotides for the
L. plantarum pdc
gene was used to screen a
B. subtilis
genomic library in order to clone the corresponding gene in this bacterium. One clone was detected with this heterologous probe, and this clone exhibited phenolic acid decarboxylase (PAD) activity. The corresponding 5-kb insertion was partially sequenced and was found to contain a 528-bp open reading frame coding for a 161-amino-acid protein exhibiting 71 and 84% identity with the
pdc
- and
fdc
-encoded enzymes, respectively. The PAD gene (
pad
) is transcriptionally regulated by
p
-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. The
pad
gene was overexpressed constitutively in
Escherichia coli
, and the stable purified enzyme was characterized. The difference in substrate specificity between this PAD and other PADs seems to be related to a few differences in the amino acid sequence. Therefore, this novel enzyme should facilitate identification of regions involved in catalysis and substrate specificity.
Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion
Authors: Xiao Rong , Kisaalita William S.
Published: December 2019

Major pyoverdines from
Pseudomonas fluorescens
2-79 (Pf-B),
P. aeruginosa
ATCC 15692 (Pa-C), and
P. putida
ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe
2+
-pyoverdine than for Fe
3+
-pyoverdine. At pH 7.4, about 90% of Fe
3+
was bound by pyoverdine Pa-C after 24 h whereas Fe
2+
was bound by the pyoverdine completely in only 5 min. The possibility that Fe
2+
underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe
2+
concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe
2+
with pyoverdine in the presence of 8-hydroxyquinoline, an Fe
3+
-specific chelating agent, resulted in the formation of a Fe
3+
-hydroxyquinoline complex, suggesting that the iron in the Fe
2+
-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.
Anaerobic Degradation of Hexadecan-2-one by a Microbial Enrichment Culture under Sulfate-Reducing Conditions
Authors: Hirschler Agnes , Rontani Jean-Francois , Raphel Danielle , Matheron Robert , Bertrand Jean-Claude
Published: December 2019

A microbial enrichment culture from marine sediment was able to grow on hexadecan-2-one as the sole source of carbon and energy under sulfate-reducing conditions. Oxidation of the ketone involved carboxylation reactions and was coupled to sulfide production. This enrichment culture also grew on 6,10,14-trimethylpentadecan-2-one.
DNA Sequence Similarity between California Isolates of
Cryptosporidium parvum
Authors: Pereira Maria das Graças C. , Atwill Edward R. , Crawford Melissa R. , Lefebvre Rance B.
Published: December 2019

We evaluated whether nucleic acid amplification with primers specific for
Cryptosporidium parvum
followed by automated DNA sequence analysis of the PCR amplicons could differentiate between California isolates of
C. parvum
obtained from livestock, humans, and feral pigs. Almost complete sequence identity existed among the livestock isolates and between the livestock and human isolates. DNA sequences from feral pig isolates differed from those from livestock and humans by 1.0 to 1.2%. The reference sequence obtained by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688–694, 1991.) differed from California isolates of
C. parvum
by 1.8 to 3.2%. These data suggest that DNA sequence analysis of the amplicon of Laxer et al. does not allow for differentiation between various strains of
C. parvum
or that our collection of isolates obtained from various hosts from across California was limited to one strain of
C. parvum.
A Gene System for Glucitol Transport and Metabolism in
Clostridium beijerinckii
NCIMB 8052
Authors: Tangney Martin , Brehm John K. , Minton Nigel P. , Mitchell Wilfrid J.
Published: December 2019

The
gutD
gene of
Clostridium beijerinckii
NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an
Escherichia coli gutD
mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order
gutA1 gutA2 orfX gutB gutD
were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from
E. coli
, while the remaining ORF (
orfX
) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which
gutD
was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The
gutA1
and
gutA2
genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by
gutB
. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the
gutB
gene. The
gut
genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the
gut
genes were not expressed until glucose was depleted.
A Deficiency in Aspartate Biosynthesis in
Lactococcus lactis
subsp.
lactis
C2 Causes Slow Milk Coagulation
Authors: Wang Hua , Yu Weizhu , Coolbear Tim , O’Sullivan Dan , McKay Larry L.
Published: December 2019

A mutant of fast milk-coagulating (Fmc
+
)
Lactococcus lactis
subsp.
lactis
C2, designated
L. lactis
KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc
−
) phenotype. When the amino acid requirements of
L. lactis
C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc
+
phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.
Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan
Authors: Yamamoto Hiroyuki , Hiraishi Akira , Kato Kenji , Chiura Hiroshi X. , Maki Yonosuke , Shimizu Akira
Published: December 2019

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the
Aquifex-Hydrogenobacter
complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the
Aquifex-Hydrogenobacter
complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.
Two New
Mycobacterium
Strains and Their Role in Toluene Degradation in a Contaminated Stream
Authors: Tay Stephen T.-L. , Hemond Harold F. , Polz Martin F. , Cavanaugh Colleen M. , Dejesus Indhira , Krumholz Lee R.
Published: December 2019

Two toluene-degrading strains, T103 and T104, were isolated from rock surface biomass in a freshwater stream contaminated with toluene. The strains exhibit different capacities for degradation of toluene and other aromatic compounds and have characteristics of the genus
Mycobacterium
. Both are aerobic, rod-shaped, gram-positive, nonmotile, and acid-alcohol fast and produce yellow pigments. They have mainly straight-chain saturated and monounsaturated fatty acids with 10 to 20 carbon atoms and large amounts of tuberculostearic acid that are typical of mycobacteria. Fatty acid analyses indicate that T103 and T104 are different mycobacterial strains that are related at the subspecies level. Their identical 16S rDNA sequences are most similar to
Mycobacterium aurum
and
Mycobacterium komossense
, and they constitute a new species of fast-growing mycobacteria. Ecological studies reveal that toluene contamination has enriched for toluene-degrading bacteria in the epilithic microbial community. Strains T103 and T104 play only a small role in toluene degradation in the stream, although they are present in the habitat and can degrade toluene. Other microorganisms are consequently implicated in the biodegradation.
Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems
Authors: Lebaron Philippe , Parthuisot Nathalie , Catala Philippe
Published: December 2019

Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4′,6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.
Sensitive and Rapid Detection of Viable
Giardia
Cysts and
Cryptosporidium parvum
Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR
Authors: Kaucner Christine , Stinear Timothy
Published: December 2019

We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable
Cryptosporidium parvum
oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable
C. parvum
oocysts,
Giardia
cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable
Giardia
cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable
C. parvum
oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable
Cryptosporidium
spp. in four samples by IF microscopy (14%), suggesting that
Cryptosporidium
species other than
C. parvum
were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.
Use of Inducible Feedback-Resistant
N
-Acetylglutamate Synthetase (
argA
) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered
Escherichia coli
K-12 Strains
Authors: Rajagopal B. S. , DePonte Joseph , Tuchman Mendel , Malamy Michael H.
Published: December 2019

The goal of this work was to construct
Escherichia coli
strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered
E. coli
strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of
N
-acetylglutamate synthetase (NAGS), the product of the
argA
gene and the first enzyme in the pathway of arginine biosynthesis in
E. coli
. To circumvent this inhibition, the
argA
genes from
E. coli
mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “
arg
boxes,” which titrate the ArgR repressor protein, with or without the
E. coli carAB
genes encoding carbamyl phosphate synthetase and the
argI
gene for ornithine transcarbamylase. The free arginine production rates of “
arg
-derepressed”
E. coli
cells overexpressing plasmid-encoded
carAB
,
argI
, and fbr
argA
genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt)
argA
. The expression system with fbr
argA
produced 7- to 35-fold more arginine than a system overexpressing
carAB
and
argI
genes on a plasmid in a strain with a wt
argA
gene on the chromosome. The arginine biosynthetic capacity of
arg
-derepressed DH5α strains with plasmids containing only the fbr
argA
gene was similar to that of cells with plasmids also containing the
carAB
and
argI
genes. Plasmids containing wt or fbr
argA
were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr
argA
mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.