bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of
Euryarchaea
Strains in the Guts and Food-Soil of the Soil-Feeding Termite
Cubitermes fungifaber
across Different Soil Types
Authors: Donovan S. E. , Purdy K. J. , Kane M. D. , Eggleton P.
Published: July 2004

Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder
Cubitermes fungifaber
Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived
Methanomicrobiales
cluster, to
Methanobrevibacter
and, surprisingly, to the haloalkaliphile
Natronococcus
. Soil clones clustered with
Methanosarcina
,
Methanomicrococcus
, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.
Isolation and Characterization of
Micromonospora
Phage ΦHAU8 and Development into a Phasmid
Authors: Li Xiaohua , Zhou Xiufen , Deng Zixin
Published: July 2004

ΦHAU8, a temperate
Micromonospora
phage, which is capable of infecting
Micromonospora
sp. strains 40027 and A-M-01, was isolated. The ΦHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. ΦHAU8 was most stable at 4°C in Difco nutrient broth within a pH range of 6 to 12. ΦHAU8 plaque formation on
Micromonospora
sp. strain 40027 was optimal with 32 mM Ca
2+
and 30 mM Mg
2+
. A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a λ cosmid vector in
Escherichia coli
and as a phage in
Micromonospora
sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that ΦHAU8 DNA integrates into the 500-kb AseI fragment of
Micromonospora
sp. strain 40027.
Serotypes and Virulence Gene Profiles of Shiga Toxin-Producing
Escherichia coli
Strains Isolated from Feces of Pasture-Fed and Lot-Fed Sheep
Authors: Djordjevic Steven P. , Ramachandran Vidiya , Bettelheim Karl A. , Vanselow Barbara A. , Holst Peter , Bailey Graham , Hornitzky Michael A.
Published: July 2004

Shiga toxin-producing
Escherichia coli
(STEC) strains possessing genes for enterohemolysin (
ehxA
) and/or intimin (
eae
), referred to here as complex STEC (cSTEC), are more commonly recovered from the feces of humans with hemolytic uremic syndrome and hemorrhagic colitis than STEC strains that do not possess these accessory virulence genes. Ruminants, particularly cattle and sheep, are recognized reservoirs of STEC populations that may contaminate foods destined for human consumption. We isolated cSTEC strains from the feces of longitudinally sampled pasture-fed sheep, lot-fed sheep maintained on diets comprising various combinations of silage and grain, and sheep simultaneously grazing pastures with cattle to explore the diversity of cSTEC serotypes capable of colonizing healthy sheep. A total of 67 cSTEC serotypes were isolated, of which 21 (31.3%), mainly isolated from lambs, have not been reported. Of the total isolations, 58 (86.6%) were different from cSTEC serotypes isolated from a recent study of longitudinally sampled healthy Australian cattle (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol.
68:
6439-6445, 2002). Our data suggest that cSTEC serotypes O5:H
−
, O75:H8, O91:H
−
, O123:H
−
, and O128:H2 are well adapted to colonizing the ovine gastrointestinal tract, since they were the most prevalent serotypes isolated from both pasture-fed and lot-fed sheep. Collectively, our data show that Australian sheep are colonized by diverse cSTEC serotypes that are rarely isolated from healthy Australian cattle.
Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast
Yarrowia lipolytica
Authors: Mlíčková Kateřina , Roux Emeline , Athenstaedt Karin , d'Andrea Sabine , Daum Günther , Chardot Thierry , Nicaud Jean-Marc
Published: July 2004

Yarrowia lipolytica
contains five acyl-coenzyme A oxidases (Aox), encoded by the
POX1
to
POX5
genes, that catalyze the limiting step of peroxisomal β-oxidation. In this study, we analyzed morphological changes of
Y. lipolytica
growing in an oleic acid medium and the effect of
POX
deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the
POX
genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Δ
pox2
Δ
pox3
Δ
pox5
), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.
Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by
Agrobacterium tumefaciens
BY6 Requires an Epimerase and a Stereoselective C-N Lyase
Authors: Cokesa Z̆eljko , Knackmuss Hans-Joachim , Rieger Paul-Gerhard
Published: July 2004

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain
Agrobacterium tumefaciens
BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe
2+
-, Mg
2+
-, and Ca
2+
-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn
2+
-IDS were rather scant and very slow on Cu
2+
-IDS. Growth and turnover experiments with
A. tumefaciens
BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating
d
-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.
Temporal and Spatial Profiles of Chitinase Expression by Norway Spruce in Response to Bark Colonization by
Heterobasidion annosum
Authors: Hietala Ari M. , Kvaalen Harald , Schmidt Axel , Jøhnk Nina , Solheim Halvor , Fossdal Carl G.
Published: July 2004

Pathogen colonization and transcript levels of three host chitinases, putatively representing classes I, II, and IV, were monitored with real-time PCR after wounding and bark infection by
Heterobasidion annosum
in 32-year-old trees of Norway spruce (
Picea abies
) with low (clone 409) or high (clone 589) resistance to this pathogen. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. At 14 days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for clone 589 but had progressed further into the host tissue in clone 409. Transcript levels of the class II and IV chitinases increased after wounding or inoculation, but the transcript level of the class I chitinase declined after these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in clone 589 than in similar sites in clone 409 3 days after inoculation. This difference was even more pronounced 2 to 6 mm away from the inoculation point, where no infection was yet established, and suggests that the clones differ in the rate of chitinase-related signal perception or transduction. At 14 days after inoculation, these transcript levels were higher in clone 409 than in clone 589, suggesting that the massive upregulation of class II and IV chitinases after the establishment of infection comes too late to reduce or prevent pathogen colonization.
Relationship between
gyrA
Mutations and Quinolone Resistance in
Flavobacterium psychrophilum
Isolates
Authors: Izumi Shotaro , Aranishi Futoshi
Published: July 2004

Flavobacterium psychrophilum
is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of
F. psychrophilum
are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27
F. psychrophilum
strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from
gyrA
nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [
Escherichia coli
numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in
F. psychrophilum
.
Genetic Characterization and Transmission Cycles of
Cryptosporidium
Species Isolated from Humans in New Zealand
Authors: Learmonth James J. , Ionas George , Ebbett Kim A. , Kwan Errol S.
Published: July 2004

Little is known about the genetic characteristics, distribution, and transmission cycles of
Cryptosporidium
species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing
Cryptosporidium
oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall,
Cryptosporidium hominis
accounted for 47% of the isolates, with the remaining 53% being the
C. parvum
bovine genotype. A difference, however, was observed between the
Cryptosporidium
species from rural and urban isolates, with
C. hominis
dominant in the urban region, whereas the
C. parvum
bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel
Cryptosporidium
sp., which on DNA sequence analysis showed a close relationship with
C. canis
, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.
Toxic and Nontoxic
Microcystis
Colonies in Natural Populations Can Be Differentiated on the Basis of rRNA Gene Internal Transcribed Spacer Diversity
Authors: Janse Ingmar , Kardinaal W. Edwin A. , Meima Marion , Fastner Jutta , Visser Petra M. , Zwart Gabriel
Published: July 2004

Assessing
and predicting bloom dynamics and toxin production by
Microcystis
requires analysis of toxic and nontoxic
Microcystis
genotypes in natural communities. We show that
genetic differentiation of
Microcystis
colonies based on rRNA
internal transcribed spacer (ITS) sequences provides an adequate basis
for recognition of microcystin producers. Consequently, ecological
studies of toxic and nontoxic cyanobacteria are now possible through
studies of rRNA ITS genotypic diversity in isolated cultures or
colonies and in natural communities. A total of 107
Microcystis
colonies were isolated from 15 lakes in Europe and
Morocco, the presence of microcystins in each colony was examined by
matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS
denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE
analysis of amplified ITSa and ITSc fragments, yielding supplementary
resolution (I. Janse et al., Appl. Environ. Microbiol.
69:
6634-6643, 2003), the colonies could be differentiated into
59 classes. Microcystin-producing and non-microcystin-producing
colonies ended up in different classes. Sequences from the rRNA ITS of
representative strains were congruent with the classification based on
DGGE and confirmed the recognition of microcystin producers on the
basis of rRNA ITS. The rRNA ITS sequences also confirmed
inconsistencies reported for
Microcystis
identification based
on morphology. There was no indication for geographical restriction of
strains, since identical sequences originated from geographically
distant lakes. About 28% of the analyzed colonies gave rise to
multiple bands in DGGE profiles, indicating either aggregation of
different colonies, or the occurrence of sequence differences between
multiple operons. Cyanobacterial community profiles from two Dutch
lakes from which colonies had been isolated showed different relative
abundances of genotypes between bloom stages and between the water
column and surface scum. Although not all bands in the community
profiles could be matched with isolated colonies, the profiles suggest
a dominance of nontoxic colonies, mainly later in the season and in
scums.
Control of
Listeria monocytogenes
in a Biofilm by Competitive-Exclusion Microorganisms
Authors: Zhao Tong , Doyle Michael P. , Zhao Ping
Published: July 2004

Biofilms from drains in food processing facilities with a recent history of no detectable
Listeria monocytogenes
in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to
L. monocytogenes
, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to
L. monocytogenes
were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10
3
CFU of
L. monocytogenes
/ml and 10
5
CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of
L. monocytogenes
growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as
Enterococcus durans
(six isolates),
Lactococcus lactis
subsp.
lactis
(two isolates), and
Lactobacillus plantarum
(one isolate). The anti-
L. monocytogenes
activity of these isolates was evaluated in biofilms of
L. monocytogenes
on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (
E. durans
strain 152 and
L. lactis
subsp.
lactis
strain C-1-92) were highly inhibitory to
L. monocytogenes
(growth inhibition of >5 log
10
CFU of
L. monocytogenes
/cm
2
). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control
L. monocytogenes
in biofilms at environmental temperatures of 4 to 37°C.
Characterization of
Salmonella enterica
Serovar Typhimurium from Marine Environments in Coastal Waters of Galicia (Spain)
Authors: Martinez-Urtaza Jaime , Liebana Ernesto , Garcia-Migura Lourdes , Perez-Piñeiro Pelayo , Saco Montserrat
Published: July 2004

Twenty-three
Salmonella enterica
serovar Typhimurium isolates from marine environments were characterized by phage typing, pulsed-field gel electrophoresis (PFGE) analysis, plasmid analysis, and antibiotic resistance, and the distribution of the different types in the coastal waters were subsequently analyzed. Five phage types were identified among the isolates (PT41, PT135, PT99, DT104, and DT193). PT135 isolates were exclusively detected during the winter months from 1998 to 2000, whereas DT104 and PT41 isolates were detected exclusively in the summer months from 2000 to 2002. XbaI PFGE analysis revealed 9 PFGE types, and plasmid profiling identified 8 plasmid types (with 1 to 6 plasmids) among the isolates. Only three isolates presented multidrug resistance to antibiotics. Two DT104 isolates were resistant to 8 and 7 antibiotics (profiles ACCeFNaSSuT and ACeFNeSSuT), whereas a PT193 isolate presented resistance to 6 antibiotics (profile ACFSSu). In addition, four PT41 isolates were resistant to a single antibiotic. The detection of multidrug-resistant phage types DT104 and DT193 in shellfish emphasizes the importance of monitoring the presence of
Salmonella
in routine surveillance of live bivalve molluscs.
Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from
Pseudomonas
sp. Strain ATCC 29352
Authors: Bhushan Bharat , Halasz Annamaria , Spain Jim C. , Hawari Jalal
Published: July 2004

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C
6
H
6
N
12
O
12
), a future-generation high-energy explosive, is biodegradable by
Pseudomonas
sp. strain FA1 and
Agrobacterium
sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from
Pseudomonas
sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min
−1
mg of protein
−1
under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M − H]
−
at 345 Da, corresponding to an empirical formula of C
6
H
6
N
10
O
8
, produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M − H]
−
at 381 Da corresponding to an empirical formula of C
6
H
10
N
10
O
10
. The latter was a hydrated product of the metabolite C
6
H
6
N
10
O
8
with addition of two H
2
O molecules, as confirmed by tests using
18
O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.
A Single Species,
Micromonas pusilla
(Prasinophyceae), Dominates the Eukaryotic Picoplankton in the Western English Channel
Authors: Not Fabrice , Latasa Mikel , Marie Dominique , Cariou Thierry , Vaulot Daniel , Simon Nathalie
Published: July 2004

The class Prasinophyceae (Chlorophyta) contains several photosynthetic picoeukaryotic species described from cultured isolates. The ecology of these organisms and their contributions to the picoeukaryotic community in aquatic ecosystems have received little consideration. We have designed and tested eight new 18S ribosomal DNA oligonucleotide probes specific for different Prasinophyceae clades, genera, and species. Using fluorescent in situ hybridization associated with tyramide signal amplification, these probes, along with more general probes, have been applied to samples from a marine coastal site off Roscoff (France) collected every 2 weeks between July 2000 and September 2001. The abundance of eukaryotic picoplankton remained high (>10
3
cells ml
−1
) during the sampling period, with maxima in summer (up to 2 × 10
4
cells ml
−1
), and a single green algal species,
Micromonas pusilla
(Prasinophyceae), dominated the community all year round. Members of the order Prasinococcales and the species
Bathycoccus prasinos
(Mamiellales) displayed sporadic occurrences, while the abundances of all other
Prasinophyceae
groups targeted remained negligible.
Structural Characterization of Lignin during
Pinus taeda
Wood Treatment with
Ceriporiopsis subvermispora
Authors: Guerra Anderson , Mendonça Régis , Ferraz André , Lu Fachuang , Ralph John
Published: July 2004

Pinus taeda
wood chips were biotreated with
Ceriporiopsis subvermispora
under solid-state fermentation for periods varying from 15 to 90 days. Milled wood lignins extracted from sound and biotreated wood samples were characterized by wet-chemical and spectroscopic techniques. Treatment of the lignins by derivatization followed by reductive cleavage (DFRC) made it possible to detect DFRC monomers and dimers that are diagnostic of the occurrence of arylglycerol-β-
O
-aryl and β-β, β-5, β-1, and 4-
O
-5 units in the lignin structure. Quantification of these DFRC products indicated that β-
O
-aryl cleavage was a significant route for lignin biodegradation but that β-β, β-5, β-1, and 4-
O
-5 linkages were more resistant to the biological attack. The amount of aromatic hydroxyls did not increase with the split of β-
O
-4 linkages, suggesting that the β-
O
-4 cleavage products remain as quinone-type structures as detected by UV and visible spectroscopy. Nuclear magnetic resonance techniques also indicated the formation of new substructures containing nonoxygenated, saturated aliphatic carbons (CH
2
and CH
3
) in the side chains of lignins extracted from biotreated wood samples.
Replication of the Endosymbiotic Bacterium
Blochmannia floridanus
Is Correlated with the Developmental and Reproductive Stages of Its Ant Host
Authors: Wolschin Florian , Hölldobler Bert , Gross Roy , Zientz Evelyn
Published: July 2004

The dynamics of replication of the intracellular endosymbiotic bacterium
Blochmannia floridanus
was determined during the larval development of its host ant
Camponotus floridanus
by real-time quantitative PCR. The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants). In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly. In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal. An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species
C. herculeanus
and
C. sericeiventris
, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes.
Diversity and Dynamics of a North Atlantic Coastal
Vibrio
Community
Authors: Thompson Janelle R. , Randa Mark A. , Marcelino Luisa A. , Tomita-Mitchell Aoy , Lim Eelin , Polz Martin F.
Published: July 2004

Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains. Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation. A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period. Quantitative PCR (QPCR) with primers specific for the genus
Vibrio
was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE).
Vibrio
populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations. Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction. It further showed that CDCE populations corresponded in most cases to ∼98% rRNA similarity groups and suggested that the abundance of these follows temperature trends. Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples. Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend.
CO
2
- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different
Listeria monocytogenes
Strains
Authors: Jydegaard-Axelsen Anne-Marie , Høiby Poul Erik , Holmstrøm Kim , Russell Nicholas , Knøchel Susanne
Published: July 2004

Although carbon dioxide (CO
2
) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen
Listeria monocytogenes
is characterized by its ability to grow in high CO
2
concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N
2
, and 100% CO
2
. The CO
2
-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant
L. monocytogenes
LO28 responded in the same manner to CO
2
as to other anaerobic, slightly acidic environments (100% N
2
, pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive
L. monocytogenes
strain EGD responded differently to CO
2
and N
2
at the same pH. In a separate experiment with
L. monocytogenes
412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO
2
-containing atmospheres. Together, our findings demonstrate that the CO
2
-response is a partly strain-dependent complex mechanism. The possible links between the CO
2
-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.
Use of the Riboflavin Synthase Gene (
ribC
) as a Model for Development of an Essential Gene Disruption and Complementation System for
Haemophilus influenzae
Authors: Saeed-Kothe Amna , Yang Wei , Mills Scott D.
Published: July 2004

We have developed a system for rapid and reliable assessment of gene essentiality in
Haemophilus influenzae
Rd strain KW20. We constructed two “suicide” complementation vectors (pASK5 and pASK6) containing 5′ and 3′ regions of the nonessential
ompP1
gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of
H. influenzae
with the complementation constructs directs chromosomal integration of a gene of interest into the
ompP1
locus, where the strong, constitutive
ompP1
promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (
ribC
), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of
ribC
for survival of
H. influenzae
Rd strain KW20 and demonstrated that a complementing copy of
ribC
placed under control of the
ompP1
promoter reverses the lethal phenotype of a strain with
ribC
deleted.
Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid
Codakia orbicularis
(Bivalvia: Lucinidae)
Authors: Duplessis Melinda R. , Ziebis Wiebke , Gros Olivier , Caro Audrey , Robidart Julie , Felbeck Horst
Published: July 2004

The large tropical lucinid clam
Codakia orbicularis
has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid
Lucinoma aequizonata
showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of
C. orbicularis
use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.
Effect of Substratum Surface Chemistry and Surface Energy on Attachment of Marine Bacteria and Algal Spores
Authors: Ista Linnea K. , Callow Maureen E. , Finlay John A. , Coleman Sarah E. , Nolasco Aleece C. , Simons Robin H. , Callow James A. , Lopez Gabriel P.
Published: July 2004

Two series of self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold were used to systematically examine the effects of varying substratum surface chemistry and energy on the attachment of two model organisms of interest to the study of marine biofouling, the bacterium
Cobetia marina
(formerly
Halomonas marina
) and zoospores of the alga
Ulva linza
(formerly
Enteromorpha linza
). SAMs were formed on gold-coated glass slides from solutions containing mixtures of methyl- and carboxylic acid-terminated alkanethiols and mixtures of methyl- and hydroxyl-terminated alkanethiols.
C. marina
attached in increasing numbers to SAMs with decreasing advancing water contact angles (θ
AW
), in accordance with equation-of-state models of colloidal attachment. Previous studies of
Ulva
zoospore attachment to a series of mixed methyl- and hydroxyl-terminated SAMs showed a similar correlation between substratum θ
AW
and zoospore attachment. When the hydrophilic component of the SAMs was changed to carboxylate, however, the profile of attachment of
Ulva
was significantly different, suggesting that a more complex model of interfacial energetics is required.
Epidemic Clone I-Specific Genetic Markers in Strains of
Listeria monocytogenes
Serotype 4b from Foods
Authors: Yildirim Suleyman , Lin Wen , Hitchins Anthony D. , Jaykus Lee-Ann , Altermann Eric , Klaenhammer Todd R. , Kathariou Sophia
Published: July 2004

Listeria monocytogenes
contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by
L. monocytogenes
detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of
L. monocytogenes
obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of
L. monocytogenes
(epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.
New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples
Authors: Gueimonde Miguel , Tölkkö Satu , Korpimäki Teemu , Salminen Seppo
Published: July 2004

The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 10
4
cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.
Chloromethane-Dependent Expression of the
cmu
Gene Cluster of
Hyphomicrobium chloromethanicum
Authors: Borodina Elena , McDonald Ian R. , Murrell J. Colin
Published: July 2004

The methylotrophic bacterium
Hyphomicrobium chloromethanicum
CM2 can utilize chloromethane (CH
3
Cl) as the sole carbon and energy source. Previously genes
cmuB
,
cmuC
,
cmuA
, and
folD
were shown to be essential for the growth of
Methylobacterium chloromethanicum
on CH
3
Cl. These CH
3
Cl-specific genes were subsequently detected in
H. chloromethanicum
. Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH
3
Cl metabolism in
H. chloromethanicum
. New developments in genetic manipulation of
Hyphomicrobium
are presented in this study. An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into
H. chloromethanicum
to generate stable CH
3
Cl-negative mutants. Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of
Hyphomicrobium
. A reliable and reproducible selection procedure for screening of CH
3
Cl utilization-negative mutants has also been developed. Mutational inactivation of
cmuB
,
cmuC
, or
hutI
resulted in strains that were unable to utilize CH
3
Cl or to express the CH
3
Cl-dependent polypeptide CmuA. Reverse transcription-PCR analysis indicated that
cmuB
,
cmuC
,
cmuA
,
fmdB
,
paaE
,
hutI
, and
metF
formed a single
cmuBCA
-
metF
operon and were coregulated and coexpressed in
H. chloromethanicum
. This finding led to the conclusion that, in
cmuB
and
cmuC
mutants, impaired expression of
cmuA
was likely to be due to a polar effect of the defective gene (
cmuB
or
cmuC
) located upstream (5′) of
cmuA
. The detrimental effect of mutation in
hutI
on the upstream (5′)-located
cmuA
is not clear but indicated that all the genes located within the
cmuBCA
-
metF
operon are coordinately expressed. Expression of the
cmuBCA
-
metF
transcript was also shown to be strictly CH
3
Cl inducible and was not repressed by the alternative C
1
substrate methanol. Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected
hutI
and
metF
genes located 3′ of the
paaE
gene in
H. chloromethanicum
. MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from
M. chloromethanicum
. Mutational and transcriptional analysis data indicated that, in
H. chloromethanicum
, CH
3
Cl is metabolized via a corrinoid-specific (
cmuA
) and tetrahydrofolate-dependent (
metF
,
purU
,
folD
) methyltransfer system.
Regulation of
Vibrio anguillarum empA
Metalloprotease Expression and Its Role in Virulence
Authors: Denkin Steven M. , Nelson David R.
Published: July 2004

Atlantic salmon (
Salmo salar
) were challenged with
Vibrio anguillarum
strains M93Sm and NB10 and
empA
null mutants M99 and NB12. Both wild types were virulent when administered by intraperitoneal (i.p.) injection or anal intubation. NB12 was avirulent via either route of infection. M99 virulence was attenuated when delivered by intubation, but fully virulent by i.p. injection. Northern blot analysis revealed
empA
expression in M93Sm and NB10 cells incubated in mucus, while incubation in Luria-Bertani broth plus 2% NaCl (LB20) induced
empA
expression only in NB10. Nucleotide differences between M93Sm and NB10
empA
sequences were found in regions located 207 and 229 bp upstream of the
empA
translational start. Reverse transcription-PCR and 5′ rapid amplification of cDNA ends revealed the
empA
transcriptional start site 85 bp upstream of the translational start for both strains. A putative σ
S
-dependent promoter was identified upstream of the transcriptional start in both strains. Site-directed mutagenesis was used to create
rpoS
mutants of M93Sm and NB10. Neither
rpoS
mutant exhibited protease activity. Since
empA
is expressed during stationary phase, the effects of conditioned medium on protease activity were examined. M99 conditioned LB20 supernatants stimulated protease activity in NB10 while allowing M93Sm to produce protease in LB20. Neither acyl homoserine lactones nor AI-2 induced protease activity. Conditioned LB20 supernatant from a
V. anguillarum luxS
mutant caused a more rapid induction of protease activity in wild-type cells. Our data show that expression of
empA
is differentially regulated in
V. anguillarum
strains NB10 and M93Sm and requires σ
S
, quorum-sensing molecules, and gastrointestinal mucus.
Host-AdaptedCryptosporidiumspp. in Canada Geese (Branta canadensis)
Authors: Zhou Ling , Kassa Hailu , Tischler Monica L. , Xiao Lihua
Published: July 2004

The prevalence and distribution ofCryptosporidiumspp. in the fecal droppings of the free-living waterfowl Canada geese were examined at 13 sites in Ohio and Illinois. On the basis of the analysis of the small-subunit rRNA gene by PCR, followed by restriction fragment length polymorphism analysis and DNA sequencing, 49 (23.4%) of 209 fecal specimens collected from 10 sites (76.9%) were positive forCryptosporidiumspp. The following fiveCryptosporidiumspecies and genotypes were identified:Cryptosporidiumgoose genotype I (in 36 specimens),Cryptosporidiumgoose genotype II (in 9 specimens),Cryptosporidiumduck genotype (in 1 specimen),Cryptosporidium parvum(in 4 specimens), andC. hominis(in 2 specimens).Cryptosporidiumgoose genotype I was the most prevalent parasite and was found at all fiveCryptosporidium-positive sites in Ohio and at four of five positive sites in Illinois, followed byCryptosporidiumgoose genotype II, which was found at two of five positive sites in Ohio and at four of five positive sites in Illinois.Cryptosporidiumgoose genotype II was detected for the first time, and it is phylogenetically related to goose genotype I and the duck genotype. All three genotypes have not so far been reported in humans, and their pathogenicity in geese has not been determined. Only 10.2% of theCryptosporidium-positive specimens hadC. parvumandC. hominis. The results of this study indicate that Canada geese might only serve as accidental carriers of cryptosporidia infectious to humans and probably play a minor role in the animal-to-human transmission cycle of the pathogen.
Molecular Analysis of Carbon Monoxide-Oxidizing Bacteria Associated with Recent Hawaiian Volcanic Deposits
Authors: Dunfield Kari E. , King Gary M.
Published: July 2004

Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (
coxL
) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified
coxL
sequences from the OMP clade of CO oxidizers, which includes isolates such as
Oligotropha carboxidovorans
,
Mycobacterium tuberculosis
, and
Pseudomonas thermocarboxydovorans
. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to
coxL
sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.
Enzymatic Synthesis of High-Molecular-Mass Poly-γ-Glutamate and Regulation of Its Stereochemistry
Authors: Ashiuchi Makoto , Shimanouchi Kazuya , Nakamura Hisaaki , Kamei Tohru , Soda Kenji , Park Chung , Sung Moon-Hee , Misono Haruo
Published: July 2004

For the first time, we succeeded in synthesizing in vitro poly-γ-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from
Bacillus subtilis
subsp.
chungkookjang
. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be ∼7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg
2+
was essential in catalysis and that Zn
2+
additionally boosted the activity. In contrast, Fe
2+
and Ca
2+
acted as inhibitors. Mn
2+
did not apparently influence the in vitro formation of PGA.
dl
-Glutamate (
d
isomer content, 60 to 80%) apparently served as the best substrate;
d
-Glutamate was preferable to the
l
isomer as a substrate. When
d
- and
l
-glutamate were used for the reaction, the elongated chains of PGAs were composed of the
d
- and
l
-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (
dl
ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the
pgsB
,
-C
, and
-A
gene products, which are responsible for PGA production by
B. subtilis
cells, were also indispensable for enzymatic PGA synthesis.
Identification of Bacterial Populations in Dairy Wastewaters by Use of 16S rRNA Gene Sequences and Other Genetic Markers
Authors: McGarvey Jeffery A. , Miller William G. , Sanchez Susan , Stanker Larry
Published: July 2004

Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the
Firmicutes
(low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of
Clostridium lituseburense
and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of
Eubacterium tenue
, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment.