bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium
Thermotoga maritima
Grown on Glucan- and Mannan-Based Polysaccharides
Authors: Chhabra Swapnil R. , Shockley Keith R. , Ward Donald E. , Kelly Robert M.
Published: February 2003

The genome sequence of the hyperthermophilic bacterium
Thermotoga maritima
encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study,
T. maritima
was grown on a range of polysaccharides, including barley β-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 10
9
cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting β-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example,
cel74
(TM0305), a gene encoding a putative β-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for
lam16
(TM0024),
xyl10A
(TM0061),
xyl10B
(TM0070), and
cel12A
(TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The
cel12B
(TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by
man5
(TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the
cel5A
(TM1751) and
cel5B
(TM1752) genes, which encode putative intracellular, β-specific endoglucanases, were induced only when
T. maritima
was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (
M
r
, 76,900) and Cel5A (
M
r
, 37,400) were expressed in
Escherichia coli
and characterized. Man5, a
T. maritima
extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley β-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when
T. maritima
was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.
Competitive Exclusion of
Salmonella enterica
serovar Enteritidis by
Lactobacillus crispatus
and
Clostridium lactatifermentans
in a Sequencing Fed-Batch Culture
Authors: van der Wielen Paul W. J. J. , Lipman Len J. A. , van Knapen Frans , Biesterveld Steef
Published: February 2003

Competitive exclusion of
Salmonella enterica
serovar Enteritidis by a mixed culture of
Lactobacillus crispatus
and
Clostridium lactatifermentans
was studied in a sequencing fed-batch reactor mimicking the cecal ecophysiology of broiler chickens. Growth of serovar Enteritidis was inhibited by a mixed culture of
L. crispatus
and
C. lactatifermentans
at pH 5.8 but not by a monoculture of
L. crispatus
at the same pH. Moreover, experiments performed at pH 7.0 did not show growth inhibition of serovar Enteritidis.
L. crispatus
fermented lactose to lactate, and
C. lactatifermentans
fermented the lactate to acetate and propionate in a mixed culture of
L. crispatus
and
C. lactatifermentans
growing on lactose. In contrast, only lactate was produced from lactose by a monoculture of
L. crispatus
. At pH 5.8 considerable concentrations of acetate and propionate were present as undissociated acids, whereas only trace levels of undissociated lactate were present at pH 5.8 due to the low pK
a
of lactate. At pH 7.0 all three acids were present in their dissociated forms. We conclude that a mixed culture of
L. crispatus
and
C. lactatifermentans
inhibits growth of serovar Enteritidis under cecal growth conditions. The undissociated forms of acetate and propionate produced in the mixed culture inhibited the growth of serovar Enteritidis.
Antimicrobial Resistance of
Escherichia coli
O157 Isolated from Humans, Cattle, Swine, and Food
Authors: Schroeder Carl M. , Zhao Cuiwei , DebRoy Chitrita , Torcolini Jocelyn , Zhao Shaohua , White David G. , Wagner David D. , McDermott Patrick F. , Walker Robert D. , Meng Jianghong
Published: February 2003

A total of 361
Escherichia coli
O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (
n
= 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing
E. coli
(STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as
E. coli
O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.
Expression of Antisense RNA Targeted against
Streptococcus thermophilus
Bacteriophages
Authors: Sturino Joseph M. , Klaenhammer Todd R.
Published: February 2003

Antisense RNA complementary to a putative helicase gene (
hel3.1
) of a
cos
-type
Streptococcus thermophilus
bacteriophage was used to impede the proliferation of a number of
cos
-type
S. thermophilus
bacteriophages and one
pac
-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the
S. thermophilus
bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal
hel3.1
probe were sensitive to the expression of antisense
hel3.1
RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the
S. thermophilus
parental strain. When progeny were released, the burst size was reduced. Growth curves of
S. thermophilus
NCK1125, in the presence of variable levels of bacteriophage κ3, showed that antisense
hel3.1
conferred protection, even at a multiplicity of infection of approximately 1.0. When the
hel3.1
antisense RNA cassette was expressed in
cis
from the κ3-derived phage-encoded resistance (PER) plasmid pTRK690::
ori3.1
, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10
−7
and 10
−8
, beyond the resistance conferred by the PER element alone (less than 10
−6
). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of
S. thermophilus
bacteriophages.
Novel Fungitoxicity Assays for Inhibition of Germination-Associated Adhesion of
Botrytis cinerea
and
Puccinia recondita
Spores
Authors: Slawecki Richard A. , Ryan Eileen P. , Young David H.
Published: February 2003

Botrytis cinerea
and
Puccinia recondita
spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC
50
s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in
B. cinerea
inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC
50
s for inhibition of mycelial growth. Similarly, in
P. recondita
the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.
Effect of Commercial-Scale High-Temperature, Short-Time Pasteurization on the Viability ofMycobacterium paratuberculosisin Naturally Infected Cows' Milk
Authors: Grant Irene R. , Hitchings Edward I. , McCartney Alan , Ferguson Fiona , Rowe Michael T.
Published: February 2003

Raw cows' milk naturally infected withMycobacterium paratuberculosiswas pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73°C for 15 s or 25 s with and without prior homogenization (2,500 lb/in2in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested forM. paratuberculosisby immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions,M. paratuberculosiswas detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viableM. paratuberculosiswas cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular,M. paratuberculosisappeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, survivingM. paratuberculosisbacteria were isolated from milk processed by all four heat treatments, i.e., 73°C for 15 and 25 s with and without prior homogenization. On one other occasion, survivingM. paratuberculosisbacteria were isolated from an unhomogenized milk sample that had been heat treated at 73°C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect toM. paratuberculosis, but the extended 25-s holding time at 73°C was found to be no more effective at killingM. paratuberculosisthan the standard 15-s holding time. This study provides clear evidence thatM. paratuberculosisbacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.
Conversion of
l
-Leucine to Isovaleric Acid by
Propionibacterium freudenreichii
TL 34 and ITGP23
Authors: Thierry Anne , Maillard Marie-Bernadette , Yvon Mireille
Published: February 2003

Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of
Propionibacterium freudenreichii
, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of
l
-leucine, α-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced α-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal norα-hydroxyisocaproic acid was detected.
l
-Isoleucine and
l
-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that
P. frendenreichii
could play a significant role in the formation of isovaleric acid during ripening.
Characterization ofWolbachiaHost Cell Range via the In Vitro Establishment of Infections
Authors: Dobson Stephen L. , Marsland Eric J. , Veneti Zoe , Bourtzis Kostas , O'Neill Scott L.
Published: February 2003

Maternally transmitted bacteria of the genusWolbachiaare obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects ofWolbachiaresearch have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias ofWolbachiaresearch toward infections that occur in host insects that are easily reared. Here, we demonstrate thatWolbachiainfections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examinedWolbachiahost range by introducing differentWolbachiatypes into a single tissue culture. The results show that anAedes albopictus(Diptera: Culicidae) cell line can support five differentWolbachiainfection types derived fromDrosophila simulans(Diptera: Drosophilidae),Culex pipiens(Culicidae), andCadra cautella(Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the majorWolbachiaclades. As an additional examination ofWolbachiahost cell range, we demonstrated that aWolbachiastrain fromD. simulanscould be established in host insect cell lines derived fromA. albopictus,Spodoptera frugiperda(Lepidoptera: Noctuidae), andDrosophila melanogaster. These results will facilitate the development of aWolbachiastock center, permitting novel approaches for the study ofWolbachiainfections and encouragingWolbachiaresearch in additional laboratories.
Culture-Independent Analysis of Gut Bacteria: the Pig Gastrointestinal Tract Microbiota Revisited
Authors: Leser Thomas D. , Amenuvor Joanna Z. , Jensen Tim K. , Lindecrona Rikke H. , Boye Mette , Møller Kristian
Published: February 2003

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the
Bacteroides
and
Prevotella
group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the
Mycoplasma
without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.
Chemostat Approach for the Directed Evolution of Biodesulfurization Gain-of-Function Mutants
Authors: Arensdorf Joseph J. , Loomis A. Katrina , DiGrazia Philip M. , Monticello Daniel J. , Pienkos Philip T.
Published: February 2003

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of
Rhodococcus erythropolis
IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes,
dszA
and
dszC
. A transversion (G to T) in
dszC
codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.
16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis
Authors: Kawai Mako , Matsutera Eiichi , Kanda Hisashi , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao
Published: February 2003

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were
Bradyrhizobium
sp.,
Xanthomonas
sp., and
Stenotrophomonas
sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.
Listeria monocytogenes
LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases
Authors: Chavant Patrick , Martinie Brigitte , Meylheuc Thierry , Bellon-Fontaine Marie-Noëlle , Hebraud Michel
Published: February 2003

The surface physicochemical properties of
Listeria monocytogenes
LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by
L. monocytogenes
LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.
Horizontal Transfer of the Plant Virulence Gene,
nec1
, and Flanking Sequences among Genetically Distinct
Streptomyces
Strains in the Diastatochromogenes Cluster
Authors: Bukhalid R. A. , Takeuchi T. , Labeda D. , Loria R.
Published: February 2003

Evidence for the horizontal transfer of a pathogenicity island (PAI) carrying the virulence gene
nec1
and flanking sequences among
Streptomyces
strains in the Diastatochromogenes cluster is presented. Plant-pathogenic, thaxtomin-producing
Streptomyces
strains, previously classified as
S. scabiei
based on the conventionally used phenotypic characteristics, were found to be genetically distinct from the type strain of
S. scabiei
based on DNA relatedness and 16S rDNA sequence analysis. Pairwise DNA-DNA hybridizations between some of these strains and the
S. scabiei
type strain were as low as 36%, a value much below what is conventionally accepted for species identity (70%). The sequence of the
nec1
gene, however, was identical in all the
S. scabiei
and
S. scabiei
-like strains tested, irrespective of their DNA relatedness to the type strain of
S. scabiei
, their geographic origin, or the isolation host. Furthermore, a 26-kb DNA fragment including and flanking
nec1
was also conserved among these strains based on restriction and Southern analyses. These data indicate that the etiology of potato scab is more complex than previously recognized; this result has important implications for potato scab management strategies. Previous research has suggested that horizontal transfer of a PAI was the mechanism for evolution of pathogenicity in
S. acidiscabies
and
S. turgidiscabies
, species that lie outside of the Diastatochromogenes cluster. Data presented here support this model and indicate that PAI transfer also has occurred frequently in species closely related to
S. scabiei
.
Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium
Thermus thermophilus
Strain HB27
Authors: Friedrich Alexandra , Prust Christina , Hartsch Thomas , Henne Anke , Averhoff Beate
Published: February 2003

Thermus thermophilus
HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of
T. thermophilus
HB27, we performed homology searches in the partially completed
T. thermophilus
genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of
Haemophilus influenzae
, seven are similar to type IV pilus proteins of
Pseudomonas aeruginosa
or
Neisseria gonorrhoeae
(PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in
Deinococcus radiodurans
R1. Analysis of the piliation phenotype of
T. thermophilus
HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent
pil
mutants of
Thermus
, with the exception of the
pilF
mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in
T. thermophilus
HB27 are functionally linked.
Osmotically Regulated Synthesis of the Compatible Solute Ectoine in
Bacillus pasteurii
and Related
Bacillus
spp
Authors: Kuhlmann Anne U. , Bremer Erhard
Published: February 2003

By using natural-abundance
13
C-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography (HPLC) analysis we have investigated the types of compatible solutes that are synthesized de novo in a variety of
Bacillus
species under high-osmolality growth conditions. Five different patterns of compatible solute production were found among the 13
Bacillus
species we studied.
Bacillus subtilis
,
B. licheniformis
, and
B. megaterium
produced proline;
B. cereus
,
B. circulans
,
B. thuringiensis
,
Paenibacillus polymyxa
, and
Aneurinibacillus aneurinilyticus
synthesized glutamate;
B. alcalophilus
,
B. psychrophilus
, and
B. pasteurii
synthesized ectoine; and
Salibacillus
(formerly
Bacillus
)
salexigens
produced both ectoine and hydroxyectoine, whereas
Virgibacillus
(formerly
Bacillus
)
pantothenticus
synthesized both ectoine and proline. Hence, the ability to produce the tetrahydropyrimidine ectoine under hyperosmotic growth conditions is widespread within the genus
Bacillus
and closely related taxa. To study ectoine biosynthesis within the group of
Bacillus
species in greater detail, we focused on
B. pasteurii
. We cloned and sequenced its ectoine biosynthetic genes (
ectABC
). The
ectABC
genes encode the diaminobutyric acid acetyltransferase (EctA), the diaminobutyric acid aminotransferase (EctB), and the ectoine synthase (EctC). Together these proteins constitute the ectoine biosynthetic pathway, and their heterologous expression in
B. subtilis
led to the production of ectoine. Northern blot analysis demonstrated that the
ectABC
genes are genetically organized as an operon whose expression is strongly enhanced when the osmolality of the growth medium is raised. Primer extension analysis allowed us to pinpoint the osmoregulated promoter of the
B. pasteurii ectABC
gene cluster. HPLC analysis of osmotically challenged
B. pasteurii
cells revealed that ectoine production within this bacterium is finely tuned and closely correlated with the osmolality of the growth medium. These observations together with the osmotic control of
ectABC
transcription suggest that the de novo synthesis of ectoine is an important facet in the cellular adaptation of
B. pasteurii
to high-osmolarity surroundings.
Enhanced Exopolysaccharide Production by Metabolic Engineering of
Streptococcus thermophilus
Authors: Levander Fredrik , Svensson Malin , Rådström Peter
Published: February 2003

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the
galU
gene, which codes for UDP glucose pyrophosphorylase (GalU) in
Streptococcus thermophilus
LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when
galU
was overexpressed in combination with
pgmA
, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal
+
) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing
galU
in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when
pgmA
was knocked out in the Gal
+
strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal
+
strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal
+
strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.
Expression of the Xylulose 5-Phosphate Phosphoketolase Gene,
xpkA
, from
Lactobacillus pentosus
MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System
Authors: Posthuma Clara C. , Bader Rechien , Engelmann Roswitha , Postma Pieter W. , Hengstenberg Wolfgang , Pouwels Peter H.
Published: February 2003

Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene,
xpkA
, from
Lactobacillus pentosus
MD363 are described.
xpkA
encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of
xpkA
in
Escherichia coli
led to an increase in XpkA activity, while an
xpkA
knockout mutant of
L. pentosus
lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that
xpkA
encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from
Bifidobacterium lactis
and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of
xpkA
in
L. pentosus
was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.
Detection and Quantification of
Pfiesteria piscicida
by Using the Mitochondrial Cytochrome
b
Gene
Authors: Zhang Huan , Lin Senjie
Published: February 2003

Mitochondrial cytochrome
b
was isolated from the dinoflagellate
Pfiesteria piscicida
, and the utility of the gene for species identification was examined. One of the primer sets designed was shown to be highly specific for
P. piscicida
. A time step PCR protocol was used to demonstrate the potential of this primer set for quantification of this species.
Simple Procedure for Rapid Identification of
Vibrio cholerae
from the Aquatic Environment
Authors: Choopun Nipa , Louis Valérie , Huq Anwar , Colwell Rita R.
Published: February 2003

Biochemical tests commonly used to screen for
Vibrio cholerae
in environmental samples were evaluated, and we found that a combination of alkaline peptone enrichment followed by streaking on thiosulfate citrate bile salts sucrose agar and testing for arginine dihydrolase activity and esculin hydrolysis was an effective rapid technique to screen for aquatic environmental
V. cholerae
. This technique provided 100% sensitivity and ≥70% specificity.
Quantitative relations between exposure to respirable quartz and risk of silicosis
Authors: Buchanan D , Miller B G , Soutar C A
Published: February 2003

Aims:To reanalyse exposure-response data from a Scottish colliery to gain a more detailed knowledge of the relations between exposure to quartz and risks of silicosis in coal miners, and hence inform the debate on an appropriate occupational standard for respirable quartz.

Methods:Detailed data on working times at different quartz concentrations were combined to produce exposure profiles for miners who had provided a full chest radiograph at a follow up survey. Logistic regression methods were used to model profusion of radiographic abnormalities category 2/1+, and a general exposure index was used to compare different quartz exposure measures in these models.

Results:Results in 371 men aged 50–74 indicated that cumulative quartz exposure at higher concentrations resulted in proportionally greater risks of abnormalities. One g.h.m−3of cumulative exposure at quartz concentrations greater than 2 mg.m−3was estimated to have equivalent risks to 3 g.h.m−3at lower concentrations. The timing of exposure relative to follow up appeared less important, although the study had limited power to compare different lag periods between exposure and effect.

Conclusions:Quantification of the risks of silicosis should take account of variations in quartz exposure intensity, particularly for exposure to concentrations of greater than 1 or 2 mg.m−3, even if exposure is for relatively short periods. The risks of silicosis over a working lifetime can rise dramatically with even brief exposure to such high quartz concentrations. Risk estimates are given, to inform choice of control limits.
Effect of trihalomethane exposure on fetal development
Authors: Wright J M , Schwartz J , Dockery D W
Published: February 2003

Aims:To examine the effect of trimester specific and pregnancy average total trihalomethane (TTHM) exposure on infant birth weight, low birth weight, and intrauterine growth retardation in term births, as well as gestational age and preterm delivery in all births.

Methods:Cross sectional analysis of 56 513 singleton infants born to residents of Massachusetts during 1990. City specific aggregate data were used to estimate maternal exposure to TTHM concentration; individual maternal information was used to adjust for confounding.

Results:Increased pregnancy average and second trimester TTHM exposure were associated with small for gestational age and reductions in birth weight after adjusting for potential confounding variables. Compared to ≤60 μg/l, pregnancy average TTHM exposure over 80 μg/l was associated with a 32 g reduction in birth weight. There was a 23 g reduction in birth weight in infants born to mothers exposed to greater than 80 μg/l TTHM during the second trimester. For each 20 μg/l increase in TTHM, the estimated reduction in birth was 2.8 g for pregnancy average exposure and 2.6 g for second trimester exposure. An increased risk of small for gestational age births was found for pregnancy average (odds ratio (OR) 1.14; 95% CI 1.02 to 1.26) and second trimester (OR 1.13, 95% CI 1.03 to 1.24) TTHM levels greater than 80 μg/l. There was no evidence of an association between preterm delivery and increased TTHM levels, but there were slight increases in gestational duration associated with TTHM concentrations.

Conclusions:Maternal exposure to THMs may be associated with fetal growth retardation. Our findings are consistent with most previous work, although we generally found smaller effects of TTHMs on low birth weight and intrauterine growth retardation.
Mental health of British farmers
Authors: Thomas H V , Lewis G , Thomas D Rh , Salmon R L , Chalmers R M , Coleman T J , Kench S M , Morgan-Capner P , Meadows D , Sillis M , Softley P
Published: February 2003

Aims:To estimate the prevalence of neurotic symptoms in a sample of British farmers, to investigate whether farming characteristics are associated with psychiatric morbidity, and to test the hypothesis that British farmers have a higher prevalence of depression and thoughts of life not worth living than the British household population.

Methods:A total of 425 farmers from Hereford, Norwich, and Preston completed the Revised Clinical Interview Schedule (CIS-R) by computer between March and July 1999. The comparison cohort consisted of 9830 private householders aged 16–64 from the Office of Population Censuses and Surveys National Psychiatric Morbidity Surveys of Great Britain carried out in 1993 in which the CIS-R was administered. All analyses used the commands developed specifically for survey data available in Stata version 6.0.

Results:Taking a threshold of an overall score of 12 or more on the CIS-R, only 6% of farmers reported clinically relevant psychiatric morbidity. Psychiatric morbidity was not significantly associated with farm type or size in this study. Farmers reported a lower prevalence of psychiatric morbidity than the general population but were more likely to report thinking that life is not worth living, particularly after the low prevalence of psychiatric morbidity had been taken into account (odds ratio 2.56, 95% CI 1.39 to 4.69). When restricting the comparison to only rural or semirural householders, this increased risk was even more pronounced (odds ratio 3.26, 95% CI 1.51 to 7.02).

Conclusions:The relation between depression and suicidal ideation seems to be quite different among farmers and the general population and warrants further investigation. We have shown it is possible to measure mental health systematically in a sample of British farmers. This study should be repeated in the aftermath of the foot and mouth crisis.
Lymphohaematopoietic system cancer incidence in an urban area near a coke oven plant: an ecological investigation
Authors: Parodi S , Vercelli M , Stella A , Stagnaro E , Valerio F
Published: February 2003

Aims:To evaluate the incidence risk of lymphohaematopoietic cancers for the 1986–94 period in Cornigliano, a district of Genoa (Italy), where a coke oven is located a few hundred metres from the residential area.

Methods:The whole of Genoa and one of its 25 districts (Rivarolo) were selected as controls. The trend of risk around the coke oven was evaluated via Stone’s method, while the geographic pattern of such risks across the Cornigliano district was evaluated by computing full Bayes estimates of standardised incidence ratio (FBE-SIR).

Results:In males, elevated relative risks (RR) were observed for all lymphohaematopoietic cancers (RR 1.7vRivarolo and 1.6vGenoa), for NHL (RR 2.4vRivarolo and 1.7vGenoa), and for leukaemia (RR 2.4vRivarolo and 1.9vGenoa). In females, statistically non-significant RR were observed. In males no excess of risk was found close to the coke oven. In females, a rising risk for NHL was observed approaching the plant, although statistical significance was not reached, while the risk for leukaemia was not evaluable due to the small number of cases. Analysis of the geographic pattern of risk suggested the presence of a cluster of NHL in both sexes in the eastern part of the district, where a foundry had been operational until the early 1980s. A cluster of leukaemia cases was observed in males in a northern part of the area, where no major sources of benzene seemed to be present.

Conclusions:The estimated risks seem to be slightly or not at all related to the distance from the coke oven. The statistically significant higher risks observed in males for NHL and leukaemia, and the clusters of leukaemia in males and of NHL in both sexes deserve further investigations in order to trace the exposures associated with such risks.
Coverage of work related fatalities in Australia by compensation and occupational health and safety agencies
Authors: Driscoll T , Mitchell R , Mandryk J , Healey S , Hendrie L , Hull B
Published: February 2003

Aims:To determine the levels of coverage of work related traumatic deaths by official occupational health and safety (OHS) and compensation agencies in Australia, to allow better understanding and interpretation of officially available statistics.

Methods:The analysis was part of a much larger study of all work related fatalities that occurred in Australia during the four year period 1989 to 1992 inclusive and which was based on information from coroners’ files. For the current study, State, Territory, and Commonwealth OHS and compensation agencies were asked to supply unit record information for all deaths identified by the jurisdictions as being due to non-suicide traumatic causes and which were identified by them as being work related, using whatever definitions the agencies were using at the relevant time. This information was matched to cases identified during the main study.

Results:The percentage of working deaths not covered by any agency was 34%. Only 35% of working deaths were covered by an OHS agency, while 57% were covered by a compensation agency. The OHS agencies had minimal coverage of work related deaths that occurred on the road (to workers (8%) or commuters (3%)), whereas the compensation system covered these deaths better than those of workers in incidents that occurred in a workplace (65% versus 53%). There was virtually no coverage of bystanders (less than 8%) by either type of agency. There was marked variation in the level of coverage depending on the industry, occupation, and employment status of the workers, and the type of injury event involved in the incident.

Conclusions:When using data from official sources, the significant limitations in coverage identified in this paper need to be taken into account. Future surveillance, arising from a computerised National Coroners Information System, should result in improved coverage of work related traumatic deaths in Australia.
Radiographic (ILO) readings predict arterial oxygen desaturation during exercise in subjects with asbestosis
Authors: Lee Y C G , Singh B , Pang S C , de Klerk N H , Hillman D R , Musk A W
Published: February 2003

Background:Exercise impairment is common in subjects with asbestosis. Arterial oxygen desaturation during exercise is an important contributor to exercise limitation. The International Labour Office (ILO) classification of plain chest radiographs correlates with resting pulmonary function, but its value in predicting abnormal ventilatory responses to exercise, including desaturation, has not been explored.

Aims:To determine in subjects with asbestosis (1) if radiographic profusion scores and the extent of small irregular shadows on plain chest radiographs correlate with resting lung function and abnormal ventilatory responses to exercise; and (2) if radiographic scores add value to resting lung function tests in predicting abnormal ventilatory responses to exercise.

Methods:Thirty eight male subjects with asbestosis were included. Plain chest radiographs were read according to the ILO classification independently by three observers. All subjects underwent assessment of lung function and an incremental exercise test.

Results:Profusion scores and number of affected zones correlated significantly with the percentage predicted values of single breath diffusing capacity (DLCO), forced vital capacity (FVC), and total lung capacity (TLC). Arterial oxygen desaturation occurred in 29% of the subjects. The severity of desaturation correlated significantly with profusion and the number of affected zones. The combined use of number of affected zones, FEV1/FVC ratio and DLCO predicted desaturation during exercise with an explained variance of 41%. VO2max was significantly related only to DLCO but was not predicted by the ILO score.

Conclusion:Arterial oxygen desaturation correlated with the profusion and extent of parenchymal abnormality on chest radiographs. The addition of morphological indices to physiological measurements is valuable for predicting oxygen desaturation during exercise but not for VO2max. Refinement of the radiographic scoring system and the addition of more sophisticated imaging techniques may further improve the predictive power.
Volcanic ash and respiratory symptoms in children on the island of Montserrat, British West Indies
Authors: Forbes L , Jarvis D , Potts J , Baxter P J
Published: February 2003

Background:In July 1995 the volcano on the West Indian island of Montserrat erupted after being inactive for several hundred years. Since then, clouds of ash have been released intermittently from the volcano. Some of this ash is <10 μm in diameter and therefore respirable. Concerns were raised that the particles might cause respiratory problems.

Aims:To evaluate whether ashfalls had any effect on the respiratory health of children in Montserrat.

Methods:A survey of asthma diagnoses, respiratory symptoms, exercise induced bronchoconstriction (EIB), and current and previous exposure to volcanic ash was carried out in schools in Montserrat during February 1998.

Results:Questionnaire information was available for nearly 80% of the 443 children on the school rolls. The prevalence of wheeze symptoms in the previous 12 months was 18% in children aged 12 years and under and 16% in children aged 13 years and over. In children aged 12 and under, the prevalence of wheeze was greater in those who had ever been heavily or moderately exposed to volcanic ash compared with the group who had only ever been exposed to low levels (wheeze in last 12 months: odds ratio (OR) 4.30; wheeze ever: OR 3.45). The prevalence of EIB in 8–12 year olds was about four times higher in those who were currently heavily exposed to volcanic ash (OR 3.85) compared to those currently exposed to low levels.

Conclusions:Volcanic ash emissions adversely affected the respiratory health of Montserrat children. The findings emphasise the need to limit exposures of children to volcanic ash and ensure the appropriate management of respiratory symptoms.
Memory one month after acute carbon monoxide intoxication: a prospective study
Authors: Deschamps D , Géraud C , Julien H , Baud F J , Dally S
Published: February 2003

Background:Serious delayed neuropsychological sequelae may complicate carbon monoxide intoxication. The existence of minor manifestations, especially memory disturbances, is not well documented.

Aims:To study several memory functions after carbon monoxide intoxication.

Methods:In a prospective study, 32 poisoned patients without risk factors for cognitive disturbances were compared to 32 paired control subjects one month after acute carbon monoxide intoxication (blood carbon monoxide concentration at least 1.0 mmol/l), who had been treated with standard conventional therapy. Psychometric tests included Buschke’s verbal memory testing, verbal digit span, Corsi’s visuospatial span, reaction times, Stroop’s colours decoding test, and verbal fluency test.

Results:(1) Memory functions in poisoned subjects were not worse than in the control group and were even better in some areas: learning, word recall, and quality of learning by Buschke’s verbal memory testing. Attention was also better in the patients, in whom visual reaction time was shorter than in controls. (2) Results of several memory functions—quality of learning and immediate visual memory—were positively correlated with the initial carbon monoxide level.

Conclusions:In a highly selected subset of patients devoid of risk factors for memory impairment, memory, objectively evaluated by psychometric testing, was not worse one month after carbon monoxide intoxication in patients undergoing standard treatment than in paired control subjects.