bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering of Primary Carbon Metabolism for Improved Antibiotic Production in
Streptomyces lividans
Authors: Butler Michael J. , Bruheim Per , Jovetic Srdjan , Marinelli Flavia , Postma Pieter W. , Bibb Mervyn J.
Published: September 2002

Deletions were made in
Streptomyces lividans
in either of two genes (
zwf1
and
zwf2
) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Δ
zwf
mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (
devB
) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both
zwf
genes from the
devB
mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.
Relationship between Bacterial Community Composition and Bottom-Up versus Top-Down Variables in Four Eutrophic Shallow Lakes
Authors: Muylaert Koenraad , Van der Gucht Katleen , Vloemans Nele , Meester Luc De , Gillis Moniek , Vyverman Wim
Published: September 2002

Bacterial community composition was monitored in four shallow eutrophic lakes during one year using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified prokaryotic rDNA genes. Of the four lakes investigated, two were of the clearwater type and had dense stands of submerged macrophytes while two others were of the turbid type characterized by the occurrence of phytoplankton blooms. One turbid and one clearwater lake had high nutrient levels (total phosphorus, >100 μg liter
−1
) while the other lakes had relatively low nutrient levels (total phosphorus, <100 μg liter
−1
). For each lake, seasonal changes in the bacterial community were related to bottom-up (resources) and top-down (grazers) variables by using canonical correspondence analysis (CCA). Using an artificial model dataset to which potential sources of error associated with the use of relative band intensities in DGGE analysis were added, we found that preferential amplification of certain rDNA genes over others does not obscure the relationship between bacterial community composition and explanatory variables. Besides, using this artificial dataset as well as our own data, we found a better correlation between bacterial community composition and explanatory variables by using relative band intensities compared to using presence/absence data. While bacterial community composition was related to phytoplankton biomass in the high-nutrient lakes no such relation was found in the low-nutrient lakes, where the bacterial community is probably dependent on other organic matter sources. We used variation partitioning to evaluate top-down regulation of bacterial community composition after bottom-up regulation has been accounted for. Using this approach, we found no evidence for top-down regulation of bacterial community composition in the turbid lakes, while grazing by ciliates and daphnids (
Daphnia
and
Ceriodaphnia
) was significantly related to changes in the bacterial community in the clearwater lakes. Our results suggest that in eutrophic shallow lakes, seasonality of bacterial community structure is dependent on the dominant substrate source as well as on the food web structure.
Growth at Low Ammonium Concentrations and Starvation Response as Potential Factors Involved in Niche Differentiation among Ammonia-Oxidizing Bacteria
Authors: Bollmann Annette , Bär-Gilissen Marie-José , Laanbroek Hendrikus J.
Published: September 2002

In nature, ammonia-oxidizing bacteria have to compete with heterotrophic bacteria and plants for limiting amounts of ammonium. Previous laboratory experiments conducted with
Nitrosomonas europaea
suggested that ammonia-oxidizing bacteria are weak competitors for ammonium. To obtain a better insight into possible methods of niche differentiation among ammonia-oxidizing bacteria, we carried out a growth experiment at low ammonium concentrations with
N. europaea
and the ammonia oxidizer G5-7, a close relative of
Nitrosomonas oligotropha
belonging to
Nitrosomonas
cluster 6a, enriched from a freshwater sediment. Additionally, we compared the starvation behavior of the newly enriched ammonia oxidizer G5-7 to that of
N. europaea
. The growth experiment at low ammonium concentrations showed that strain G5-7 was able to outcompete
N. europaea
at growth-limiting substrate concentrations of about 10 μM ammonium, suggesting better growth abilities of the ammonia oxidizer G5-7 at low ammonium concentrations. However,
N. europaea
displayed a more favorable starvation response. After 1 to 10 weeks of ammonium deprivation,
N. europaea
became almost immediately active after the addition of fresh ammonium and converted the added ammonium within 48 to 96 h. In contrast, the regeneration time of the ammonia oxidizer G5-7 increased with increasing starvation time. Taken together, these results provide insight into possible mechanisms of niche differentiation for the ammonia-oxidizing bacteria studied. The
Nitrosomonas
cluster 6a member, G5-7, is able to grow at ammonium concentrations at which the growth of
N. europaea
, belonging to
Nitrosomonas
cluster 7, has already ceased, providing an advantage in habitats with continuously low ammonium concentrations. On the other hand, the ability of
N. europaea
to become active again after longer periods of starvation for ammonium may allow better exploitation of irregular pulses of ammonium in the environment.
Differences in Attachment of
Salmonella enterica
Serovars and
Escherichia coli
O157:H7 to Alfalfa Sprouts
Authors: Barak J. D. , Whitehand L. C. , Charkowski A. O.
Published: September 2002

Numerous
Salmonella enterica
and
Escherichia coli
O157:H7 outbreaks have been associated with contaminated sprouts. We examined how
S. enterica
serovars,
E. coli
serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts. Growth on and adherence to sprouts were not significantly different among different serovars of
S. enterica
, but all
S. enterica
serovars grew on and adhered to alfalfa sprouts significantly better than
E. coli
O157:H7.
E. coli
O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of
S. enterica
remained attached per sprout.
S. enterica
Newport adhered to 3-day-old sprouts as well as
Pantoea agglomerans
and 10-fold more than
Pseudomonas putida
and
Rahnella aquatilis
, whereas the growth rates of all four strains throughout seed sprouting were similar.
S. enterica
Newport and plant-associated bacteria adhered 10- to 1,000-fold more than
E. coli
O157:H7; however, three of four other
E. coli
serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than
E. coli
O157:H7 and as well as the
Pseudomonas
and
Rahnella
strains. Therefore, attachment to alfalfa sprouts among
E. coli
serotypes is variable, and nonpathogenic strains of
E. coli
to be used as surrogates for the study of pathogenic
E. coli
may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.
Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from
Rhodococcus rhodochrous
Authors: Seth-Smith Helena M. B. , Rosser Susan J. , Basran Amrik , Travis Emma R. , Dabbs Eric R. , Nicklin Steve , Bruce Neil C.
Published: September 2002

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant.
Rhodococcus rhodochrous
strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene,
xplA
, which is found in a gene cluster with an adrenodoxin reductase homologue,
xplB
. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.
Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria
Authors: Forster Scott , Snape Jason R. , Lappin-Scott Hilary M. , Porter Jonathan
Published: September 2002

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.
Resuscitation of
Salmonella enterica
Serovar Typhimurium and Enterohemorrhagic
Escherichia coli
from the Viable but Nonculturable State by Heat-Stable Enterobacterial Autoinducer
Authors: Reissbrodt R. , Rienaecker I. , Romanova J. M. , Freestone P. P. E. , Haigh R. D. , Lyte M. , Tschäpe H. , Williams P. H.
Published: September 2002

Salmonella enterica
serovar Typhimurium and enterohemorrhagic
Escherichia coli
were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for
Salmonella
only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of
S. enterica
serovar Typhimurium
rpoS
mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the σ
s
subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of
Citrobacter freundii
and
Enterobacter agglomerans
.
Anaerobic Oxidation of Arsenite in Mono Lake Water and by a Facultative, Arsenite-Oxidizing Chemoautotroph, Strain MLHE-1
Authors: Oremland Ronald S. , Hoeft Shelley E. , Santini Joanne M. , Bano Nasreen , Hollibaugh Ryan A. , Hollibaugh James T.
Published: September 2002

Arsenite [As(III)]-enriched anoxic bottom water from Mono Lake, California, produced arsenate [As(V)] during incubation with either nitrate or nitrite. No such oxidation occurred in killed controls or in live samples incubated without added nitrate or nitrite. A small amount of biological As(III) oxidation was observed in samples amended with Fe(III) chelated with nitrolotriacetic acid, although some chemical oxidation was also evident in killed controls. A pure culture, strain MLHE-1, that was capable of growth with As(III) as its electron donor and nitrate as its electron acceptor was isolated in a defined mineral salts medium. Cells were also able to grow in nitrate-mineral salts medium by using H
2
or sulfide as their electron donor in lieu of As(III). Arsenite-grown cells demonstrated dark
14
CO
2
fixation, and PCR was used to indicate the presence of a gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase. Strain MLHE-1 is a facultative chemoautotroph, able to grow with these inorganic electron donors and nitrate as its electron acceptor, but heterotrophic growth on acetate was also observed under both aerobic and anaerobic (nitrate) conditions. Phylogenetic analysis of its 16S ribosomal DNA sequence placed strain MLHE-1 within the haloalkaliphilic
Ectothiorhodospira
of the γ-
Proteobacteria
. Arsenite oxidation has never been reported for any members of this subgroup of the
Proteobacteria
.
Replacement of Trifluoroacetic Acid with HCl in the Hydrophobic Purification Steps of Pediocin PA-1: a Structural Effect
Authors: Gaussier Hélène , Morency Hélène , Lavoie Marc C. , Subirade Muriel
Published: September 2002

Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1.
Sequencing and Transcriptional Analysis of the Chlorite Dismutase Gene of
Dechloromonas agitata
and Its Use as a Metabolic Probe
Authors: Bender Kelly S. , O'Connor Susan M. , Chakraborty Romy , Coates John D. , Achenbach Laurie A.
Published: September 2002

The dismutation of chlorite into chloride and O
2
represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene
cld
was isolated and sequenced from the perchlorate-reducing bacterium
Dechloromonas agitata
strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified
D. agitata
chlorite dismutase enzyme. The predicted translation product of the
D. agitata cld
gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the −10 promoter region of the
D. agitata cld
gene. Northern blot analysis indicated that the
cld
gene was transcriptionally up-regulated when
D. agitata
cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a
D. agitata cld
probe demonstrated the conservation of the
cld
gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.
Identification of a Hydratase and a Class II Aldolase Involved in Biodegradation of the Organic Solvent Tetralin
Authors: Hernáez M. J. , Floriano B. , Ríos J. J. , Santero E.
Published: September 2002

Two new genes whose products are involved in biodegradation of the organic solvent tetralin were identified. These genes, designated
thnE
and
thnF
, are located downstream of the previously identified
thnD
gene and code for a hydratase and an aldolase, respectively. A sequence comparison of enzymes similar to ThnE showed the significant similarity of hydratases involved in biodegradation pathways to 4-oxalocrotonate decarboxylases and established four separate groups of related enzymes. Consistent with the sequence information, characterization of the reaction catalyzed by ThnE showed that it hydrated a 10-carbon dicarboxylic acid. The only reaction product detected was the enol tautomer, 2,4-dihydroxydec-2-ene-1,10-dioic acid. The aldolase ThnF showed significant similarity to aldolases involved in different catabolic pathways whose substrates are dihydroxylated dicarboxylic acids and which yield pyruvate and a semialdehyde. The reaction products of the aldol cleavage reaction catalyzed by ThnF were identified as pyruvate and the seven-carbon acid pimelic semialdehyde. ThnF and similar aldolases showed conservation of the active site residues identified by the crystal structure of 2-dehydro-3-deoxy-galactarate aldolase, a class II aldolase with a novel reaction mechanism, suggesting that these similar enzymes are class II aldolases. In contrast, ThnF did not show similarity to 4-hydroxy-2-oxovalerate aldolases of other biodegradation pathways, which are significantly larger and apparently are class I aldolases.
Prevalence and Characterization of Non-O157 Shiga Toxin-Producing
Escherichia coli
on Carcasses in Commercial Beef Cattle Processing Plants
Authors: Arthur Terrance M. , Barkocy-Gallagher Genevieve A. , Rivera-Betancourt Mildred , Koohmaraie Mohammad
Published: September 2002

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing
Escherichia coli
(STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic
E. coli hlyA
,
eae
, and at least one
stx
gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.
Multiplex Fluorogenic Real-Time PCR for Detection and Quantification ofEscherichia coliO157:H7 in Dairy Wastewater Wetlands
Authors: Ibekwe A. Mark , Watt Pamela M. , Grieve Catherine M. , Sharma Vijay K. , Lyons Steven R.
Published: September 2002

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such asEscherichia colifrom urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantifyE. coliO157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae)gene ofE. coliO157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33E. coliO157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity ofE. coliO157:H7 DNA. A similar correlation was observed between theCTand number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10−5pg ofE. coliO157:H7 DNA ml−1equivalent to approximately 6.4 × 103CFU ofE. coliO157:H7 ml−1based on plate counts was determined. Quantification ofE. coliO157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 104CFU g−1.E. coliO157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g−1with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination ofE. coliO157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.
Biotransformation of 2-Benzoxazolinone and 2-Hydroxy-1,4-Benzoxazin-3-one by Endophytic Fungi Isolated from
Aphelandra tetragona
Authors: Zikmundová M. , Drandarov K. , Bigler L. , Hesse M. , Werner C.
Published: September 2002

The biotransformation of the phytoanticipins 2-benzoxazolinone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) by four endophytic fungi isolated from
Aphelandra tetragona
was studied. Using high-performance liquid chromatography-mass spectrometry, several new products of acylation, oxidation, reduction, hydrolysis, and nitration were identified.
Fusarium sambucinum
detoxified BOA and HBOA to
N
-(2-hydroxyphenyl)malonamic acid.
Plectosporium tabacinum
,
Gliocladium cibotii
, and
Chaetosphaeria
sp. transformed HBOA to 2-hydroxy-
N
-(2-hydroxyphenyl)acetamide,
N
-(2-hydroxyphenyl)acetamide,
N
-(2-hydroxy-5-nitrophenyl)acetamide,
N
-(2-hydroxy-3-nitrophenyl)acetamide, 2-amino-3
H
-phenoxazin-3-one, 2-acetylamino-3
H
-phenoxazin-3-one, and 2-(
N
-hydroxy)acetylamino-3
H
-phenoxazin-3-one. BOA was not degraded by these three fungal isolates. Using 2-hydroxy-
N
-(2-hydroxyphenyl)[
13
C
2
]acetamide, it was shown that the metabolic pathway for HBOA and BOA degradation leads to
o
-aminophenol as a key intermediate.
Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations
Authors: Mills David A. , Johannsen Eric A. , Cocolin Luca
Published: September 2002

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (∼30°C) and ambient (∼20°C) temperatures. Fermentations at both temperatures possessed similar populations ofSaccharomyces,Hanseniaspora,Pichia,Metschnikowia,Kluyveromyces, andCandidaspecies. However, higher populations of non-Saccharomycesyeasts persisted in ambient-temperature fermentations, withCandidaand, to a lesser extent,Kluyveromycesspecies remaining long after the fermentation was dominated bySaccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed aHanseniaspora osmophilapopulation not identified in the plating analysis. rRNA analysis also indicated a large population (>106cells per ml) of a nonculturableCandidastrain in the high-temperature fermentation. Monoculture analysis of theCandidaisolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations ofCandida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.
Bacillus thuringiensis
in Fecal Samples from Greenhouse Workers after Exposure to
B. thuringiensis
-Based Pesticides
Authors: Jensen Gert B. , Larsen Preben , Jacobsen Bodil L. , Madsen Bodil , Smidt Lasse , Andrup Lars
Published: September 2002

In a study of occupational exposure to
Bacillus thuringiensis
, 20 exposed greenhouse workers were examined for
Bacillus cereus
-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of
B. thuringiensis
, genes for and production of
B. cereus
enterotoxins, and positivity for
cry11
as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of
B. thuringiensis
in the fecal samples (10
2
to 10
3
CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of
B. thuringiensis
in the fecal samples.
Diversity of Endophytic Bacterial Populations and Their Interaction withXylella fastidiosain Citrus Plants
Authors: Araújo Welington L. , Marcon Joelma , Maccheroni Walter , van Elsas Jan Dirk , van Vuurde Jim W. L. , Azevedo João Lúcio
Published: September 2002

Citrus variegated chlorosis (CVC) is caused byXylella fastidiosa, a phytopathogenic bacterium that can infect allCitrus sinensiscultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis asBacillus pumilus,Curtobacterium flaccumfaciens,Enterobacter cloacae,Methylobacteriumspp. (includingMethylobacterium extorquens,M. fujisawaense,M. mesophilicum,M. radiotolerans, andM. zatmanii),Nocardiasp.,Pantoea agglomerans, andXanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species ofMethylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolatedC. flaccumfacienssignificantly more frequently from asymptomatic plants than from those with symptoms of CVC whileP. agglomeranswas frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency ofC. flaccumfaciensin asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.
Phenotypic and Genotypic Characterization of AvianEscherichia coliO86:K61 Isolates Possessing a Gamma-Like Intimin
Authors: La Ragione R. M. , McLaren I. M. , Foster G. , Cooley W. A. , Woodward M. J.
Published: September 2002

Escherichia coliO86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identifiedE. coliO86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34E. coliO86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for theeaeAgene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacingE. coli.
Growth-Dependent Stable Carbon Isotope Fractionation by Basidiomycete Fungi: δ
13
C Pattern and Physiological Process
Authors: Henn Matthew R. , Gleixner Gerd , Chapela Ignacio H.
Published: September 2002

We grew 11 basidiomycetes in axenic culture to characterize their physiological capacities to fractionate stable C isotopes. Generally, δ
13
C values of the fungal biomass were (i) enriched in
13
C relative to the growth medium, (ii) variable among the isolates, and (iii) dependent on the growth rate and growth stage of the fungi. We found a multiphasic dynamic of fractionation for
Cryptoporus volvatus
and
Marasmius androsaceus
during various growth stages. The first phase, P1, corresponded to the exponential growth stage and was characterized by an increasing enrichment in
13
C content of the fungal biomass relative to the growth medium ranging between 4.6 and 6.9‰. The second phase, P2, exhibited a continual depletion in
13
C of the fungal biomass, with the δ
13
C values of the fungal biomass asymptotically returning to the δ
13
C value of the growth medium at inoculation. The expression of the various fractionation phases was dependent on the amount of low-concentration micronutrients and growth factors added to the growth medium. The onset of P2 occurred at reduced concentrations of these elements. All of the sugars in the growth medium (sucrose, maltose, and glucose) were utilized for growth, indicating that the observed fractionation was not an artifact derived from the preferential use of
13
C-rich maltose, which was found at low concentrations in the growth medium. In this study, we establish a framework with which to explore the impact of physiological fractionations by fungal interfaces on natural distributions of stable C isotopes.
Accumulation of 2-Aminophenoxazin-3-one-7-Carboxylate during Growth of
Pseudomonas putida
TW3 on 4-Nitro-Substituted Substrates Requires 4-Hydroxylaminobenzoate Lyase (PnbB)
Authors: Hughes Michelle A. , Baggs Michael J. , al-Dulayymi Juma'a , Baird Mark S. , Williams Peter A.
Published: September 2002

During growth of
Pseudomonas putida
strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a λ
max
of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned
pnb
structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site.
Widespread and Persistent Populations of a Major New Marine Actinomycete Taxon in Ocean Sediments
Authors: Mincer Tracy J. , Jensen Paul R. , Kauffman Christopher A. , Fenical William
Published: September 2002

A major taxon of obligate marine bacteria within the order
Actinomycetales
has been discovered from ocean sediments. Populations of these bacteria (designated MAR 1) are persistent and widespread, spanning at least three distinct ocean systems. In this study, 212 actinomycete isolates possessing MAR 1 morphologies were examined and all but two displayed an obligate requirement of seawater for growth. Forty-five of these isolates, representing all observed seawater-requiring morphotypes, were partially sequenced and found to share characteristic small-subunit rRNA signature nucleotides between positions 207 and 468 (
Escherichia coli
numbering). Phylogenetic characterization of seven representative isolates based on almost complete sequences of genes encoding 16S rRNA (16S ribosomal DNA) yielded a monophyletic clade within the family
Micromonosporaceae
and suggests novelty at the genus level. This is the first evidence for the existence of widespread populations of obligate marine actinomycetes. Organic extracts from cultured members of this new group exhibit remarkable biological activity, suggesting that they represent a prolific resource for biotechnological applications.
Differential Inhibition of Mollicute Growth: an Approach to Development of Selective Media for Specific Mollicutes
Authors: Keçeli S. A. , Miles R. J.
Published: September 2002

The energy-generating pathways of
Mycoplasma
spp. are diverse. Thus, it was predicted that the ability of inhibitors of these pathways to block growth would vary among species. This prediction was tested with 14
Mycoplasma
species and potential inhibitors. The greatest differentiation among test species was obtained using fluoride, iodoacetate (IAA), β-fluoropyruvate (FP), cibacron blue (CB),
l
-citrulline, and carbonyl cyanide
m
-chlorophenylhydrazone. A range of other potential inhibitors, including
l
-arginine analogues, had little inhibitory effect on growth, and
d
-arginine was shown to be a growth substrate for arginine-hydrolyzing species. Fluoride selectively inhibited the growth of mycoplasmas that were able only to ferment sugars to lactate and/or to hydrolyze arginine. In contrast, IAA was most effective against organic acid-oxidizing species, and
l
-citrulline inhibited arginine-hydrolyzing species.
Mycoplasma verecundum
, a species for which energy sources have not been identified, was relatively resistant to FP. Similarly,
Acholeplasma laidlawii
was distinguished by its CB resistance.
Novel Cyanobacterial Biosensor for Detection of Herbicides
Authors: Shao C. Y. , Howe C. J. , Porter A. J. R. , Glover L. A.
Published: September 2002

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium,Synechocystissp. strain PCC6803, was chromosomally marked with the luciferase geneluc(from the fireflyPhotinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of thelucgene during growth ofSynechocystissp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.
Improvement of Recombinant Protein Yield by a Combination of Transcriptional Amplification and Stabilization of Gene Expression
Authors: Cebolla A. , Royo J. L. , de Lorenzo V. , Santero E.
Published: September 2002

We explored the use of a cascade circuit for heterologous gene expression that consists of a regulatory module with a salicylate-inducible system that controls the expression of a second regulator,
xylS2
, whose product is activated by common inducers. Activation and increasing the concentration of the second regulator synergistically induced heterologous genes downstream of the
Pm
promoter in the expression module. This module can be placed in multicopy vectors or in the chromosome of a host strain by means of minitransposons. Using reporter genes, we evaluated gene regulation capacity and gross production of the system with different configurations. The highest yield was obtained when the expression module was in a multicopy plasmid after a 6-h induction. However, expression modules in plasmids showed low stability after induction even with selective pressure. The chromosomal configuration had the lowest basal levels and induced levels comparable to those of plasmid configurations, resulting in accumulation of more than 10% of the total protein. Unlike the configurations in plasmids, the yield was maintained for at least 3 days even without selective pressure. In conclusion, the cascade system in the chromosome configuration is more efficient for long-term fermentation because of the great stability of the overexpressing phenotype in spite of the high levels of expression.
Molecular Analysis of a Bacterial Chitinolytic Community in an Upland Pasture
Authors: Metcalfe A. C. , Krsek M. , Gooday G. W. , Prosser J. I. , Wellington E. M. H.
Published: September 2002

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.
A Plasmid Selection System in
Lactococcus lactis
and Its Use for Gene Expression in
L. lactis
and Human Kidney Fibroblasts
Authors: Glenting Jacob , Madsen Søren M. , Vrang Astrid , Fomsgaard Anders , Israelsen Hans
Published: September 2002

We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic
Lactococcus lactis
MG1363Δthr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the
hom
-
thrB
operon) as a selective marker. Using strain MG1363Δthr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in
L. lactis
. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.
Rapid and Sensitive Enumeration of Viable Diluted Cells of Members of the Family
Enterobacteriaceae
in Freshwater and Drinking Water
Authors: Baudart Julia , Coallier Josée , Laurent Patrick , Prévost Michèle
Published: September 2002

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family
Enterobacteriaceae
in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10
8
nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.
Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment
Authors: Loy Alexander , Lehner Angelika , Lee Natuschka , Adamczyk Justyna , Meier Harald , Ernst Jens , Schleifer Karl-Heinz , Wagner Michael
Published: September 2002

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of
Desulfomicrobium
spp. in the tooth pockets and the presence of
Desulfonema
- and
Desulfomonile
-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (
dsrAB
).
Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid ofBacillus thuringiensissubsp.israelensis
Authors: Berry Colin , O'Neil Susan , Ben-Dov Eitan , Jones Andrew F. , Murphy Lee , Quail Michael A. , Holden Mathew T. G. , Harris David , Zaritsky Arieh , Parkhill Julian
Published: September 2002

The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis fromBacillus thuringiensissubsp.israelensisis presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.
Variability in
Pseudomonas aeruginosa
Lipopolysaccharide Expression during Crude Oil Degradation
Authors: Norman R. Sean , Frontera-Suau Roberto , Morris Pamela J.
Published: September 2002

Bacterial utilization of crude oil components, such as the
n
-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two
Pseudomonas aeruginosa
strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the
n
-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased
n
-alkane degradation.