bluuebunny/crossref_metadata_2025 huggingface dataset:
Dehalogenation of Haloalkanes byMycobacterium tuberculosisH37Rv and Other Mycobacteria
Authors: Jesenská Andrea , Sedlác̆ek Ivo , Damborský Jir̆í
Published: April 2009

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasiteMycobacterium tuberculosisH37Rv. The ability ofM. tuberculosisand several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells ofM. tuberculosisH37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains,Mycobacterium aviumMU1 andMycobacterium smegmatisCCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.
Sources of
Escherichia coli
in a Coastal Subtropical Environment
Authors: Solo-Gabriele Helena M. , Wolfert Melinda A. , Desmarais Timothy R. , Palmer Carol J.
Published: April 2009

Sources of
Escherichia coli
in a coastal waterway located in Ft. Lauderdale, Fla., were evaluated. The study consisted of an extensive program of field measurements designed to capture spatial and temporal variations in
E. coli
concentrations as well as experiments conducted under laboratory-controlled conditions.
E. coli
from environmental samples was enumerated by using a defined substrate technology (Colilert-18). Field sampling tasks included sampling the length of the North Fork to identify the river reach contributing high
E. coli
levels, autosampler experiments at two locations, and spatially intense sampling efforts at hot spots. Laboratory experiments were designed to simulate tidal conditions within the riverbank soils. The results showed that
E. coli
entered the river in a large pulse during storm conditions. After the storm,
E. coli
levels returned to baseline levels and varied in a cyclical pattern which correlated with tidal cycles. The highest concentrations were observed during high tide, whereas the lowest were observed at low tide. This peculiar pattern of
E. coli
concentrations between storm events was caused by the growth of
E. coli
within riverbank soils which were subsequently washed in during high tide. Laboratory analysis of soil collected from the riverbanks showed increases of several orders of magnitude in soil
E. coli
concentrations. The ability of
E. coli
to multiply in the soil was found to be a function of soil moisture content, presumably due to the ability of
E. coli
to outcompete predators in relatively dry soil. The importance of soil moisture in regulating the multiplication of
E. coli
was found to be critical in tidally influenced areas due to periodic wetting and drying of soils in contact with water bodies. Given the potential for growth in such systems,
E. coli
concentrations can be artificially elevated above that expected from fecal impacts alone. Such results challenge the use of
E. coli
as a suitable indicator of water quality in tidally influenced areas located within tropical and subtropical environments.
Documenting the Epidemiologic Patterns of Polyomaviruses in Human Populations by Studying Their Presence in Urban Sewage
Authors: Bofill-Mas Sílvia , Pina Sonia , Girones Rosina
Published: April 2009

This is the first description, to our knowledge, of the distribution of human polyomavirus and simian virus 40 (SV40) in urban sewage. Using a nested-PCR procedure, we report the detection of human polyomaviruses JC virus (JCV) and BK virus (BKV) but not SV40 in a high percentage of urban sewage samples obtained from widely divergent geographical areas in Europe and Africa. For a total of 28 samples analyzed, JCV was detected in 26, BKV was detected in 22, and none was positive for SV40. All geographical areas showed a high prevalence of these viruses with mean estimated values of JC viral particles per ml on the order of 10
3
in Barcelona (Spain) and Nancy (France) and 10
2
in Pretoria (South Africa) and Umeå (Sweden) and mean values of BK viral particles on the order of 10
2
in Pretoria and Barcelona and 10
1
in Nancy and Umeå. This compares with estimated mean values of 10
2
to 10
3
for human adenovirus that was evaluated as a control. Nucleotide sequence analysis of the amplified DNA from some of the samples is also presented and represents the sequence of the most abundant JC and BK viral strains in these samples. The nucleotide sequence of the JCV detected was also analyzed in a phylogenetic study and for genomic characterization in the regulatory region. This study has shown that human polyomaviruses are spread in high concentrations in the sewage of different geographical areas and are present in contaminated environments. The frequency and concentration of JCV detected in the environment and the absence of described animal hosts suggest that JCV may be useful as a marker for fecal pollution of anthropogenic origin. The results also support the idea previously described that the strains of JCV are closely related to the ethnic origin of the population studied. The procedure applied should also be useful in future studies of population patterns of viral excretion and as a tool in epidemiological studies for the detection of changes in the prevalence of specific viral pathogens.
Comparative Study of Pressure- and Nutrient-Induced Germination of
Bacillus subtilis
Spores
Authors: Wuytack Elke Y. , Soons Johan , Poschet Filip , Michiels Chris W.
Published: April 2009

Germination experiments with specific germination mutants of
Bacillus subtilis
, including a newly isolated mutant affected in pressure-induced germination, suggest that a pressure of 100 MPa triggers the germination cascades that are induced by the nutrient germinant alanine (Ala) and by a mixture of asparagine, glucose, fructose, and potassium ions (AGFK), by activating the receptors for alanine and asparagine, GerA and GerB, respectively. As opposed to germination at 100 MPa, germination at 600 MPa apparently shortcuts at least part of the Ala- and AGFK-induced germination pathways. Inhibitors of nutrient-induced germination (HgCl
2
and
N
α-P-tosyl-
l
-arginine methyl ester) also inhibit pressure-induced germination at 600 MPa, suggesting that germination at 600 MPa involves activation of a true physiological germination pathway and is therefore not merely a physico-chemical process in which water is forced into the spore protoplast.
A Pilot Study of Bacteriological Population Changes through Potable Water Treatment and Distribution
Authors: Norton Cheryl D. , LeChevallier Mark W.
Published: April 2009

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.
Carbon Catabolite Repression in
Lactobacillus pentosus
: Analysis of the
ccpA
Region
Authors: Mahr Kerstin , Hillen Wolfgang , Titgemeyer Fritz
Published: April 2009

The catabolite control protein CcpA is a central regulator in low-G+C-content gram-positive bacteria. It confers carbon catabolite repression to numerous genes required for carbon utilization. It also operates as a transcriptional activator of genes involved in diverse phenomena, such as glycolysis and ammonium fixation. We have cloned the
ccpA
region of
Lactobacillus pentosus. ccpA
encodes a protein of 336 amino acids exhibiting similarity to CcpA proteins of other bacteria and to proteins of the LacI/GalR family of transcriptional regulators. Upstream of
ccpA
was found an open reading frame with similarity to the
pepQ
gene, encoding a prolidase. Primer extension experiments revealed two start sites of transcription for
ccpA
. In wild-type cells grown on glucose, mRNA synthesis occurred only from the promoter proximal to
ccpA
. In a
ccpA
mutant strain, both promoters were used, with increased transcription from the distant promoter, which overlaps a presumptive CcpA binding site called
cre
(for catabolite responsive element). This suggests that expression of
ccpA
is autoregulated. Determination of the expression levels of CcpA in cells grown on repressing and nonrepressing carbon sources revealed that the amounts of CcpA produced did not change significantly, leading to the conclusion that the arrangement of two promoters may ensure constant expression of
ccpA
under various environmental conditions. A comparison of the genetic structures of
ccpA
regions revealed that lactic acid bacteria possess the gene order
pepQ-ccpA
-variable while the genetic structure in bacilli and
Staphylococcus xylosus
is
aroA-ccpA
-variable-
acuC
.
In Situ Hybridization of
Prochlorococcus
and
Synechococcus
(Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes
Authors: Worden Alexandra Z. , Chisholm Sallie W. , Binder Brian J.
Published: April 2009

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in
Prochlorococcus
, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of
Prochlorococcus
cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions.
Prochlorococcus
cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium,
Synechococcus
, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In
Synechococcus
cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (
r
= 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.
Isolation and Expression in
Escherichia coli
of
cslA
and
cslB
, Genes Coding for the Chondroitin Sulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B, Respectively, from
Flavobacterium heparinum
Authors: Tkalec Ana Lydia , Fink Dominique , Blain Françoise , Zhang-Sun Guiyi , Laliberte Maryse , Bennett D. Clark , Gu Kangfu , Zimmermann Joseph J. F. , Su Hongsheng
Published: April 2009

In medium supplemented with chondroitin sulfate,
Flavobacterium heparinum
synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC
4.2.2.5
) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from
F. heparinum
and designated
cslA
(chondroitinase AC) and
cslB
(chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of
F. heparinum
, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The
cslA
and
cslB
DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. Truncated
cslA
and
cslB
genes have been used to produce active, mature chondroitinases in the cytoplasm of
Escherichia coli
. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from
F. heparinum.
An Explosive Antisense RNA Strategy for Inhibition of a Lactococcal Bacteriophage
Authors: Walker Shirley A. , Klaenhammer Todd R.
Published: April 2009

The coding regions of six putative open reading frames (ORFs) identified near the phage φ31 late promoter and the right cohesive end (
cos
) of lactococcal bacteriophage φ31 were used to develop antisense constructs to inhibit the proliferation of phage φ31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong
Lactobacillus
P6 promoter and the T7 terminator (T
T7
) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage φ31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage φ31 origin of replication,
ori31. ori31
allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of
ori31
also lowers the burst size of phage φ31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of
ori31
and P6::anti-ORF 4H::T
T7
resulted in a threefold decrease in the EOP of phage φ31 (EOP = 0.11 ± 0.03 [mean ± standard deviation]) compared to the presence of
ori31
alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for
ori31
compared to 35 to 45% for
ori31
plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage φ31 showed that
ori31
plus anti-ORF 4H offered significant protection to
Lactococcus lactis
, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.
Lack of Host Specialization inAspergillus flavus
Authors: St. Leger Raymond J. , Screen Steven E. , Shams-Pirzadeh Bijan
Published: April 2009

Aspergillusspp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates ofAspergillus flavus,Aspergillus fumigatus, andAspergillus nidulansfor their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains ofA. flavusdid not affect nonwounded bean leaves, corn kernels, or insects at 22°C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton bolls, is produced by mostA. flavusisolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. AllA. flavusstrains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast toA. flavus, strains ofA. nidulansandA. fumigatuscould not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate thatA. flavushas parasitic attributes that are lacking inA. fumigatusandA. nidulansbut that individual strains ofA. flavusare not specialized to particular hosts.
Rhizosphere Microbial Community Structure in Relation to Root Location and Plant Iron Nutritional Status
Authors: Yang Ching-Hong , Crowley David E.
Published: April 2009

Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status.
Genetic Rearrangements of the Regions Adjacent to Genes Encoding Heat-Labile Enterotoxins (
eltAB
) of Enterotoxigenic
Escherichia coli
Strains
Authors: Schlör Stefan , Riedl Sabine , Blaß Julia , Reidl Joachim
Published: April 2009

One of the most common bacterially mediated diarrheal infections is caused by enterotoxigenic
Escherichia coli
(ETEC) strains. ETEC-derived plasmids are responsible for the distribution of the genes encoding the main toxins, namely, the heat-labile and heat-stable enterotoxins. The origins and transfer modes (intra- or interplasmid) of the toxin-encoding genes have not been characterized in detail. In this study, we investigated the DNA regions located near the heat-labile enterotoxin-encoding genes (
eltAB
) of several clinical isolates. It was found that the
eltAB
region is flanked by conserved 236- and 280-bp regions, followed by highly variable DNA sequences which consist mainly of partial insertion sequence (IS) elements. Furthermore, we demonstrated that rearrangements of the
eltAB
region of one particular isolate, which harbors an IS
91
R sequence next to
eltAB
, could be produced by a
recA
-independent but IS
91
sequence-dependent mechanism. Possible mechanisms of dissemination of IS element-associated enterotoxin-encoding genes are discussed.
Use of Green Fluorescent Protein To Tag Lactic Acid Bacterium Strains under Development as Live Vaccine Vectors
Authors: Geoffroy Marie-Claude , Guyard Cyril , Quatannens Brigitte , Pavan Sonia , Lange Marc , Mercenier Annick
Published: April 2009

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e.,
Lactococcus lactis
and
Lactobacillus plantarum
. We have constructed different expression vectors carrying the
gfp
(green fluorescent protein [GFP]) gene from the jellyfish
Aequoria victoria
, and we found that this visible marker was best expressed when placed under the control of the inducible strong
nisA
promoter from
L. lactis
. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent
L. plantarum
NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.
Transcriptional Organization and Regulation of a Polycistronic Cold Shock Operon in
Sinorhizobium meliloti
RM1021 Encoding Homologs of the
Escherichia coli
Major Cold Shock Gene
cspA
and Ribosomal Protein Gene
rpsU
Authors: O'Connell Kevin P. , Thomashow Michael F.
Published: April 2009

A homolog of the major eubacterial cold shock gene
cspA
was identified in
Sinorhizobium meliloti
RM1021 by
luxAB
reporter transposon mutagenesis. Here we further characterize the organization and regulation of this locus. DNA sequence analysis indicated that the locus includes three open reading frames (ORFs) encoding homologs corresponding to CspA, a novel 10.6-kDa polypeptide designated ORF2, and a homolog of the
Escherichia coli
ribosomal protein S21. Transcription analysis indicated that this locus produced two different-sized
cspA
-hybridizing transcripts upon cold shock, a 400-nucleotide (nt) RNA encoding
cspA
alone and a 1,000-nt transcript encoding
cspA-ORF2-rpsU
. The sizes of the transcripts agreed with the location of the transcription start site determined by primer extension and the locations of two putative transcriptional terminators. The promoter of the
cspA-ORF2-rpsU
locus had −10 and −35 elements similar to the
E. coli
ς
70
consensus promoter and, like the
cspA
locus of
E. coli
, included an AT-rich region upstream of the −35 hexamer. The promoter of the
S. meliloti cspA
locus was found to impart cold shock-induced mRNA accumulation. In addition, the 5′-untranslated region (5′ UTR) was found to increase the fold induction of
cspA
transcripts after cold shock and depressed the level of
luxAB
mRNA prior to cold shock, another feature similar to
cspA
regulation in
E. coli
. No “cold box” was identified upstream of the
S. meliloti cspA
gene, however, and there was no other obvious sequence identity between the
S. meliloti
5′ UTR and that of
E. coli
. DNA hybridization analysis indicated that outside the
cspA-ORF2-rpsU
cold shock locus there are several additional
cspA
-like genes and a second
rpsU
homolog.
Identification of Cold Shock Gene Loci in
Sinorhizobium meliloti
by Using a
luxAB
Reporter Transposon
Authors: O'Connell Kevin P. , Gustafson Ann M. , Lehmann M. Deane , Thomashow Michael F.
Published: April 2009

Using a
luxAB
reporter transposon, seven mutants of
Sinorhizobium meliloti
were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15°C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven
luxAB
mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three
rrn
operons of
S. meliloti
. Directed insertion of
luxAB
genes into each of the three
rrn
operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major
Escherichia coli
cold shock gene,
cspA
. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15°C.
Comparison of Animal Infectivity and Nucleic Acid Staining for Assessment of
Cryptosporidium parvum
Viability in Water
Authors: Neumann Norman F. , Gyürek Lyndon L. , Gammie Leslie , Finch Gordon R. , Belosevic Miodrag
Published: April 2009

Cryptosporidium parvum
oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.
Two Murine Monoclonal Antibodies against Serogroup E Salmonellae
Authors: Ng S. P. , Tsang R. S. W. , Luk J. M. C. , Ng M. H. , Im S. W. K.
Published: April 2009

A monoclonal antibody (MAb), MO15, was raised against the lipopolysaccharide antigen of an ɛ15-lysogenized serogroup E
1
Salmonella
strain. The O factor 15-specific MAb MO15, together with another serogroup E-specific MAb, can differentiate among phage lysogenization variants in serogroup E salmonellae. Their epitope specificities in relation to conventional O-antigenic structures are discussed.
Specific Detection of
Leuconostoc mesenteroides
subsp.
mesenteroides
with DNA Primers Identified by Randomly Amplified Polymorphic DNA Analysis
Authors: Moschetti Giancarlo , Blaiotta Giuseppe , Villani Francesco , Coppola Salvatore
Published: April 2009

Randomly amplified polymorphic DNA analysis using primer 239 (5′ CTGAAGCGGA 3′) was performed to characterize
Leuconostoc
sp. strains. All the strains of
Leuconostoc mesenteroides
subsp.
mesenteroides
(with the exception of two strains), two strains formerly identified as
L. gelidum
, and one strain of
Leuconostoc
showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying
L. mesenteroides
subsp.
mesenteroides
strains.
Pulsed-Electric Field Treatment Enhances the Bactericidal Action of Nisin against
Bacillus cereus
Authors: Pol Irene E. , Mastwijk Hennie C. , Bartels Paul V. , Smid Eddy J.
Published: April 2009

Vegetative cells of
Bacillus cereus
were subjected to low doses of nisin (0.06 μg/ml) and mild pulsed-electric field treatment (16.7 kV/cm, 50 pulses each of 2-μs duration). Combining both treatments resulted in a reduction of 1.8 log units more than the sum of the reductions obtained with the single treatments, indicating synergy.
Coaggregation between Aquatic Bacteria Is Mediated by Specific-Growth-Phase-Dependent Lectin-Saccharide Interactions
Authors: Rickard Alex H. , Leach Stephen A. , Buswell Clive M. , High Nicola J. , Handley Pauline S.
Published: April 2009

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of
Blastomonas natatoria
and one strain of
Micrococcus luteus
varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.
Establishment of a Polychlorinated Biphenyl-Dechlorinating Microbial Consortium, Specific for Doubly Flanked Chlorines, in a Defined, Sediment-Free Medium
Authors: Wu Qingzhong , Sowers Kevin R. , May Harold D.
Published: April 2009

Estuarine sediment from Charleston Harbor, South Carolina, was used as inoculum for the development of an anaerobic enrichment culture that specifically dechlorinates doubly flanked chlorines (i.e., chlorines bound to carbon that are flanked on both sides by other chlorine-carbon bonds) of polychlorinated biphenyls (PCBs). Dechlorination was restricted to the
para
chlorine in cultures enriched with 10 mM fumarate, 50 ppm (173 μM) 2,3,4,5-tetrachlorobiphenyl, and no sediment. Initially the rate of dechlorination decreased upon the removal of sediment from the medium. However, the dechlorinating activity was sustainable, and following sequential transfer in a defined, sediment-free estuarine medium, the activity increased to levels near that observed with sediment. The culture was nonmethanogenic, and molybdate, ampicillin, chloramphenicol, neomycin, and streptomycin inhibited dechlorination activity; bromoethanesulfonate and vancomycin did not. Addition of 17 PCB congeners indicated that the culture specifically removes double flanked chlorines, preferably in the
para
position, and does not attack
ortho
chlorines. This is the first microbial consortium shown to
para
or
meta
dechlorinate a PCB congener in a defined sediment-free medium. It is the second PCB-dechlorinating enrichment culture to be sustained in the absence of sediment, but its dechlorinating capabilities are entirely different from those of the other sediment-free PCB-dechlorinating culture, an
ortho
-dechlorinating consortium, and do not match any previously published Aroclor-dechlorinating patterns.
Sequence Analysis of the Cryptic Plasmid pMG101 from
Rhodopseudomonas palustris
and Construction of Stable Cloning Vectors
Authors: Inui Masayuki , Roh Jung Hyeob , Zahn Kenneth , Yukawa Hideaki
Published: April 2009

A 15-kb cryptic plasmid was obtained from a natural isolate of
Rhodopseudomonas palustris
. The plasmid, designated pMG101, was able to replicate in
R. palustris
and in closely related strains of
Bradyrhizobium japonicum
and phototrophic
Bradyrhizobium
species. However, it was unable to replicate in the purple nonsulfur bacterium
Rhodobacter sphaeroides
and in
Rhizobium
species. The replication region of pMG101 was localized to a 3.0-kb
Sal
I-
Xho
I fragment, and this fragment was stably maintained in
R. palustris
for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative
rep
gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based
Escherichia coli-R. palustris
shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in
R. palustris
growing under nonselective conditions. The ability of plasmid pMG101 to replicate in
R. palustris
and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria.
Distribution and Physiological Characteristics of Hyperthermophiles in the Kubiki Oil Reservoir in Niigata, Japan
Authors: Takahata Yoh , Nishijima Miyuki , Hoaki Toshihiro , Maruyama Tadashi
Published: April 2009

The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58°C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera
Thermococcus
and
Thermotoga
, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 × 10
3
to 4.6 × 10
4
cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. Dominant
Thermococcus
and
Thermotoga
spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50°C, culturable cells of
Thermococcus
sp. remained around the initial cell density for about 200 days, while those of
Thermotoga
sp. decreased exponentially to 0.01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.
Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water
Authors: McElhiney Jacqui , Drever Mathew , Lawton Linda A. , Porter Andy J.
Published: April 2009

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed inEscherichia colifor characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacteriumMicrocystis aeruginosawere assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb inE. coliis an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.
Diversity of
Aphanizomenon
flos-aquae
(Cyanobacterium) Populations along a Baltic Sea Salinity Gradient
Authors: Laamanen Maria J. , Forsström Laura , Sivonen Kaarina
Published: April 2009

Colony-forming cyanobacteria of the genus
Aphanizomenon
form massive blooms in the brackish water of the Baltic Sea during the warmest summer months. There have been recent suggestions claiming that the Baltic Sea
Aphanizomenon
species may be different from
Aphanizomenon
flos-aquae
found in lakes. In this study, we examined variability in the morphology and 16S-23S rRNA internal transcribed spacer (ITS) sequences of
A
.
flos-aquae
populations along a salinity gradient from a string of lakes to a fjord-like extension of the Baltic Sea to the open Baltic Sea. Morphological differences among the populations were negligible. We found that the Baltic Sea was dominated (25 out of 27 sequences) by one ITS1-S (shorter band of ITS 1 [ITS1]) genotype, which also was found in the lakes. The lake populations of
A
.
flos-aquae
tended to be genetically more diverse than the Baltic Sea populations. Since the lake ITS1-S genotypes of
A
.
flos-aquae
are continuously introduced to the Baltic Sea via inflowing waters, it seems that only one ITS1 genotype is able to persist in the Baltic Sea populations. The results suggest that one of the ITS1-S genotypes found in the lakes is better adapted to the conditions of the Baltic Sea and that natural selection removes most of the lake genotypes from the Baltic Sea
A
.
flos-aquae
populations.
Isolation and Functional Analysis of a Gene,
tcsB
, Encoding a Transmembrane Hybrid-Type Histidine Kinase from
Aspergillus nidulans
Authors: Furukawa Kentaro , Katsuno Yasuaki , Urao Takeshi , Yabe Tomio , Yamada-Okabe Toshiko , Yamada-Okabe Hisafumi , Yamagata Youhei , Abe Keietsu , Nakajima Tasuku
Published: April 2009

We cloned and characterized a novel
Aspergillus nidulans
histidine kinase gene,
tcsB
, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (
SLN1
), which transmits signals through the high-osmolarity glycerol response 1 (
HOG1
) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an
A. nidulans
cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted
tcsB
protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the
tcsB
cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective
sln1-ts
yeast mutant. However,
tcsB
cDNAs in which the conserved phosphorylation site His
552
residue or the phosphorelay site Asp
989
residue had been replaced failed to complement the
sln1-ts
mutant. In addition, introduction of the
tcsB
cDNA into an
sln1
Δ
sho1
Δ yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an
A. nidulans
strain lacking the
tcsB
gene (
tcsB
Δ) and examined its phenotype. However, unexpectedly, the
tcsB
Δ strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that
A. nidulans
has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.
Ethylene Production byBotrytis cinereaIn Vitro and in Tomatoes
Authors: Cristescu Simona M. , De Martinis Domenico , te Lintel Hekkert Sacco , Parker David H. , Harren Frans J. M.
Published: April 2009

A laser-based ethylene detector was used for on-line monitoring of ethylene released by the phytopathogenic fungusBotrytis cinereain vitro and in tomato fruit. Ethylene data were combined with the results of a cytological analysis of germination ofB. cinereaconidia and hyphal growth. We found that aminoethoxyvinylglycine and aminooxyacetic acid, which are competitive inhibitors of the 1-aminocyclopropane-1-carboxylic acid pathway, did not inhibit the ethylene emission byB. cinereaand that the fungus most likely produces ethylene via the 2-keto-4-methylthiobutyric acid pathway.B. cinereais able to produce ethylene in vitro, and the emission of ethylene follows the pattern that is associated with hyphal growth rather than the germination of conidia. Ethylene production in vitro depended on thel-methionine concentration added to the plating medium. Higher values and higher emission rates were observed when the concentration of conidia was increased. Compared with the ethylene released by the fungus, the infection-related ethylene produced by two tomato cultivars (cultivars Money Maker and Daniela) followed a similar pattern, but the levels of emission were 100-fold higher. The time evolution of enhanced ethylene production by the infected tomatoes and the cytological observations indicate that ethylene emission by the tomato-fungus system is not triggered by the ethylene produced byB. cinerea, although it is strongly synchronized with the growth rate of the fungus inside the tomato.
Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NO

x

) by Two Different Lithotrophs
Authors: Schmidt Ingo , Hermelink Cristian , van de Pas-Schoonen Katinka , Strous Marc , op den Camp Huub J. , Kuenen J. Gijs , Jetten Mike S. M.
Published: April 2009

The anaerobic ammonia-oxidizing activity of the planctomycete
Candidatus
“Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO
2
concentrations up to 100 ppm.
B. anammoxidans
was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO
2
, the specific ammonia oxidation activity of
B. anammoxidans
increased, and
Nitrosomonas
-like microorganisms recovered an NO
2
-dependent anaerobic ammonia oxidation activity. Addition of NO
2
to a mixed population of
B. anammoxidans
and
Nitrosomonas
induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH
4
+
g of protein
−1
h
−1
by
B. anammoxidans
and up to 1.5 mmol of NH
4
+
g of protein
−1
h
−1
by
Nitrosomonas
. The stoichiometry of the converted N compounds (NO
2
−
/NH
3
ratio) and the microbial community structure were strongly influenced by NO
2
. The combined activity of
B. anammoxidans
and
Nitrosomonas
-like ammonia oxidizers might be of relevance in natural environments and for technical applications.
Differentiation of Closely Related
Carnobacterium
Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism
Authors: Kabadjova Petia , Dousset Xavier , Le Cam Virginie , Prevost Hervé
Published: April 2009

A novel strategy for identification of
Carnobacterium
food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all
Carnobacterium
strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another.
Carnobacterium divergens
NCDO 2763
T
and
C. mobile
DSM 4849
T
generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from
C. gallinarum
NCFB 2766
T
and
C. piscicola
NCDO 2762
T
were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA
Ile
and tRNA
Ala
genes. The M-ISR included one tRNA
Ala
gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with
Hin
fI,
Taq
I, and
Hin
dIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four
Carnobacterium
species. This method is very suitable for rapid, low-cost identification of a wide variety of
Carnobacterium
species without sequencing.
Evaluation of the Heat Inactivation of
Escherichia coli
and
Lactobacillus plantarum
by Differential Scanning Calorimetry
Authors: Lee Jaesung , Kaletunç Gönül
Published: April 2009

Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and reversibility after heat treatment of transitions associated with various cellular components of
Escherichia coli
and
Lactobacillus plantarum
. The reversibility and the change in the thermal stability of individual transitions are evaluated by a second temperature scan after preheating in the DSC to various temperatures between 40 and 130°C. The viability of bacteria after a heat treatment between 55 and 70°C in the DSC is determined by both plate count and calorimetric data. The fractional viability values based on calorimetric and plate count data show a linear relationship. Viability loss and the irreversible change in DSC thermograms of pretreated whole cells are highly correlated between 55 and 70°C. Comparison of DSC scans for isolated ribosomes shows that the thermal stability of
E. coli
ribosomes is greater than that of
L. plantarum
ribosomes, consistent with the greater thermal tolerance of
E. coli
observed from viability loss and DSC scans of whole cells.