bluuebunny/crossref_metadata_2025 huggingface dataset:
Three Transporters Mediate Uptake of Glycine Betaine and Carnitine by
Listeria monocytogenes
in Response to Hyperosmotic Stress
Authors: Angelidis Apostolos S. , Smith Gary M.
Published: February 2003

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen
Listeria monocytogenes
to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in
L. monocytogenes
: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of
L. monocytogenes
10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in
L. monocytogenes
10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.
Depth Distribution of Microbial Diversity in Mono Lake, a Meromictic Soda Lake in California
Authors: Humayoun Shaheen B. , Bano Nasreen , Hollibaugh James T.
Published: February 2003

We analyzed the variation with depth in the composition of members of the domain
Bacteria
in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain
Bacteria
: α- and γ-
Proteobacteria
(6 and 10%, respectively),
Cytophaga
-
Flexibacter
-
Bacteroides
(19%), high-G+C-content gram-positive organisms (
Actinobacteria
; 25%), and low-G+C-content gram-positive organisms (
Bacillus
and
Clostridium
; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented β- and δ-
Proteobacteria
,
Verrucomicrobiales
, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (
P
= 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (
P
< 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (
P
= 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.
Bacteriocin Detection from Whole Bacteria by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Authors: Hindré Thomas , Didelot Sandrine , Le Pennec Jean-Paul , Haras Dominique , Dufour Alain , Vallée-Réhel Karine
Published: February 2003

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.
Comparison of Shiga Toxin Production by Hemolytic-Uremic Syndrome-Associated and Bovine-Associated Shiga Toxin-Producing
Escherichia coli
Isolates
Authors: Ritchie Jenny M. , Wagner Patrick L. , Acheson David W. K. , Waldor Matthew K.
Published: February 2003

There is considerable diversity among Shiga toxin (Stx)-producing
Escherichia coli
(STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.
Molecular Analyses of
Salmonella
enterica
Isolates from Fish Feed Factories and Fish Feed Ingredients
Authors: Nesse Live L. , Nordby Kerstin , Heir Even , Bergsjoe Bjarne , Vardund Traute , Nygaard Halvor , Holstad Gudmund
Published: February 2003

Isolates of the most commonly observed salmonella serovars in Norwegian fish feed factories from 1998 to 2000 (
Salmonella enterica
serovar Agona,
S
.
enterica
serovar Montevideo,
S
.
enterica
serovar Senftenberg, and
S
.
enterica
serovar Kentucky) were studied by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis and compared to isolates of the same serovars from fish feed ingredients, humans, and other sources (a total of 112 isolates). Within each serovar, a variety of distinct PFGE types (with similarity levels less than 90%) were observed in the feed ingredients and other sources, while only two distinct types of each serovar were identified in the factories. The combined results of PFGE and plasmid analyses showed that each factory harbored only a few
S
.
enterica
clones. Some of these clones persisted for at least 3 years in the factories, indicating that there was long-lasting contamination probably due to inadequate decontamination procedures.
Small, Acid-Soluble Proteins as Biomarkers in Mass Spectrometry Analysis of
Bacillu
s Spores
Authors: Hathout Yetrib , Setlow Barbara , Cabrera-Martinez Rosa-Martha , Fenselau Catherine , Setlow Peter
Published: February 2003

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different
Bacillus
spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor α/β- and γ-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (
B. cereus
T and
B. thuringiensis
subsp.
Kurstaki
) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.
A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast
Pichia pastoris
Authors: Hanquier José , Sorlet Yannick , Desplancq Dominique , Baroche Laurence , Ebtinger Marc , Lefèvre Jean-François , Pattus Franc , Hershberger Charles L. , Vertès Alain A.
Published: February 2003

We produced bovine trypsinogen in the yeast
Pichia pastoris
. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong
aox1
promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.
Effects of Alternative Dietary Substrates on Competition between Human Colonic Bacteria in an Anaerobic Fermentor System
Authors: Duncan Sylvia H. , Scott Karen P. , Ramsay Alan G. , Harmsen Hermie J. M. , Welling Gjalt W. , Stewart Colin S. , Flint Harry J.
Published: February 2003

Duplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion ofBacteroidesand a lower proportion of gram-positive anaerobes related toFaecalibacterium prausnitzii,Ruminococcus flavefaciens-Ruminococcus bromii,Eubacterium rectale-Clostridium coccoides, andEubacterium cylindroidesthan the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related toR. flavefaciens-R. bromiiandE. cylindroides.The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. TheRoseburia-related strain A2-183Fwas able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast,Roseburia intestinalisL1-82RandEubacteriumsp. strain A2-194Rsurvived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194Rwas stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.
Monitoring the Production of Aflatoxin B
1
in Wheat by Measuring the Concentration of
nor-1
mRNA
Authors: Mayer Zsuzsanna , Färber Paul , Geisen Rolf
Published: February 2003

A real-time reverse transcription-PCR system has been used to monitor the expression of an aflatoxin biosynthetic gene of
Aspergillus flavus
in wheat. Therefore, total RNA was isolated from infected wheat samples, reverse transcribed and subjected to real-time PCR. In parallel all samples were analyzed by high-pressure liquid chromatography for aflatoxin B
1
production. The primer-probe system of the real-time PCR was targeted against
nor-1
, a gene of the aflatoxin biosynthetic pathway. By application of this method the
nor-1
transcription was quantified during the course of incubation. After 4 days of incubation
nor-1
mRNA could be detected for the first time. The amount of
nor-1
mRNA increased rapidly, and the maximum was achieved after 6 days. Then, starting very slowly, the mRNA was degraded until day 8, and this was followed by a very fast degradation, reaching nondetectable levels at days 9 and 10. First traces of aflatoxin B
1
could be detected between the 5th and 6th day of incubation. The aflatoxin concentration reached its maximum after 9 days of incubation and remained constant for the whole period of observation. To ensure that differences in the
nor-1
mRNA concentration were due to different expression levels, the expression of the constitutively expressed β-tubulin gene (
benA56
) has also been monitored. The expression of
benA56
remained constant during the whole incubation time. As a parameter for fungal growth, the number of
nor-1
gene copies was determined during the course of incubation. The numbers of
nor-1
gene copies increased at the beginning of the incubation and reached a plateau at day 5. They correlate well with the viable counts albeit at a higher level.
Involvement of Calcium/Calmodulin Signaling in Cercosporin Toxin Biosynthesis byCercosporanicotianae
Authors: Chung Kuang-Ren
Published: February 2003

Cercosporin is a non-host-selective, perylenequinone toxin produced by many phytopathogenicCercosporaspecies. The involvement of Ca2+/calmodulin (CaM) signaling in cercosporin biosynthesis was investigated by using pharmacological inhibitors. The results suggest that maintaining endogenous Ca2+homeostasis is required for cercosporin biosynthesis inCercosporanicotianae. The addition of excess Ca2+to the medium slightly increased fungal growth but resulted in a reduction in cercosporin production. The addition of Ca2+chelators [EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] also reduced cercosporin production. Ca2+channel blockers exhibited a strong inhibition of cercosporin production only at higher concentrations (>2 mM). Cercosporin production was reduced greatly by Ca2+ionophores (A23187 and ionomycin) and internal Ca2+blocker [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester]. Phospholipase C inhibitors (lithium, U73122, and neomycin) led to a concentration-dependent inhibition of cercosporin biosynthesis. Furthermore, the addition of CaM inhibitors (compound 48/80, trifluoperazine, W-7, and chlorpromazine) also markedly reduced cercosporin production. In contrast to W-7, W-5, with less specificity for CaM, led to only minor inhibition of cercosporin production. The inhibitory effects of Ca2+/CaM inhibitors were partially or completely reversed by the addition of external Ca2+. As assessed with Fluo-3/AM (a fluorescent Ca2+indicator), the Ca2+content in the cytoplasm decreased significantly when fungal cultures were grown in a medium containing Ca2+/CaM antagonists, confirming the specificity of those Ca2+/CaM antagonists inC.nicotianae. Taken together, the results suggest that Ca2+/CaM signal transduction may play a pivotal role in cercosporin biosynthesis inC.nicotianae.
Development of
Sinorhizobium meliloti
Pilot Macroarrays for Transcriptome Analysis
Authors: Bergès Hélène , Lauber Emmanuelle , Liebe Carine , Batut Jacques , Kahn Daniel , de Bruijn Frans J. , Ampe Frédéric
Published: February 2003

In order to prepare for whole-genome expression analysis in
Sinorhizobium meliloti
, pilot DNA macroarrays were designed for 34 genes of known regulation. The experimental parameters assessed were the length of the PCR products, the influence of a tag at the 5′ end of the primers, and the method of RNA labeling. Variance and principal-component analysis showed that the most important nonbiological parameter was the labeling method. The sizes of PCR products were also found to be important, whereas the influence of 5′ tags was minimal. The variability between replicated spots on a membrane was found to be low. These experimental procedures were validated by analyzing the effects of microaerobic conditions on gene expression.
Characterization of a New Operon,as-48EFGH, from theas-48Gene Cluster Involved in Immunity to Enterocin AS-48
Authors: Diaz Marta , Valdivia Eva , Martínez-Bueno Manuel , Fernández Matilde , Soler-González Andrés Santos , Ramírez-Rodrigo Hilario , Maqueda Mercedes
Published: February 2003

Enterocin AS-48 is a cyclic peptide produced byEnterococcus faecalisS-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, theas-48Astructural gene andas-48B,as-48C,as-48C1,as-48D, andas-48D1genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in theBglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs,as-48Gandas-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5disruption experiments in the last genes,as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. Theas-48EFGHoperon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T2and T3, in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T3could be regulated, because in JH2-2(pAM401EH) transformants, T3was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for theas-48D1gene for immunity against AS-48.
Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant
Escherichia
coli
Strains
Authors: Ignatova Z. , Mahsunah A. , Georgieva M. , Kasche V.
Published: February 2003

Using periplasmic penicillin amidase (PA) from
Escherichia
coli
ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant
E
.
coli
expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (

k
d

) and transport (

k
t

) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain
E
.
coli
BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants

k
d

and

k
t

. The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different
E
.
coli
host strains. Furthermore, we established that simultaneous coexpression of the OmpT
pac
gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the
kil
gene (killing protein). The primary effects of the
kil
gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.
Aminopeptidase C of
Aspergillus niger
Is a Novel Phenylalanine Aminopeptidase
Authors: Basten Daniëlle E. J. W. , Dekker Peter J. T. , Schaap Peter J.
Published: February 2003

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from
Aspergillus niger
(CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine
para
-nitroanilide (F-
p
NA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either
p
NA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze
N
-acetylalanine-
p
NA, a substrate for acyl-peptide hydrolases.
Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various
Streptomyces avermitilis
Strains
Authors: Hwang Yong-Soon , Kim Eung-Soo , Biró Sándor , Choi Cha-Yong
Published: February 2003

To isolate a gene for stimulating avermectin production, a genomic library of
Streptomyces avermitilis
ATCC 31267 was constructed in
Streptomyces lividans
TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type
S. avermitilis
was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of
Streptomyces coelicolor
A3(2). The
orfX
gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of
orfX
revealed that this gene was intrinsically involved in the stimulation of avermectin production in
S. avermitilis
.
Molecular Characterization of
tet
(M) Genes in
Lactobacillus
Isolates from Different Types of Fermented Dry Sausage
Authors: Gevers Dirk , Danielsen Morten , Huys Geert , Swings Jean
Published: February 2003

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc
r
) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the
tet
genes, conferring tetracycline resistance, that they carry. With the (GTG)
5
-PCR DNA fingerprinting technique, the Tc
r
lactic acid bacterial isolates were identified as
Lactobacillus plantarum
,
L. sakei
subsp.
carnosus
,
L. sakei
subsp.
sakei
,
L. curvatus
, and
L. alimentarius
and typed to the intraspecies level. For a selection of 24 Tc
r
lactic acid bacterial isolates displaying unique (GTG)
5
-PCR fingerprints,
tet
genes were determined by means of PCR, and only
tet
(M) was detected. Restriction enzyme analysis with
Acc
I and
Sca
I revealed two different
tet
(M) allele types. This grouping was confirmed by partial sequencing of the
tet
(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with
tet
(M) genes previously reported in
Staphylococcus aureus
MRSA 101 and in
Neisseria meningitidis
, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained
tet
(M)-carrying plasmids. In addition to the
tet
(M) gene, one isolate also contained an
erm
(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the
tet
(M) genes were not located on transposons of the Tn
916
/Tn
1545
family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse
Lactobacillus
strains from different types of fermented meat products can be a host for plasmid-borne
tet
genes.
Enhancement of Population Size of a Biological Control Agent and Efficacy in Control of Bacterial Speck of Tomato through Salicylate and Ammonium Sulfate Amendments
Authors: Ji Pingsheng , Wilson Mark
Published: February 2003

Sodium salicylate and ammonium sulfate were applied to leaf surfaces along with suspensions of the biological control agents
Pseudomonas syringae
Cit7(pNAH7), which catabolizes salicylate, and Cit7, which does not catabolize salicylate, to determine whether enhanced biological control of bacterial speck of tomato could be achieved. Foliar amendment with salicylate alone significantly enhanced the population size and the efficacy of Cit7(pNAH7), but not of Cit7, on tomato leaves. Application of ammonium sulfate alone did not result in enhanced population size or biological control efficacy of either Cit7(pNAH7) or Cit7; however, when foliar amendments with both sodium salicylate and ammonium sulfate were applied, a trend toward further increases in population size and biological control efficacy of Cit7(pNAH7) was observed. This study demonstrates the potential of using a selective carbon source to improve the efficacy of a bacterial biological control agent in the control of a bacterial plant disease and supports previous conclusions that the growth of
P. syringae
in the phyllosphere is primarily carbon limited and secondarily nitrogen limited.
Enhanced Production of Recombinant Proteins in
Escherichia coli
by Filamentation Suppression
Authors: Jeong Ki Jun , Lee Sang Yup
Published: February 2003

During growth of high-cell-density cultures of
Escherichia coli
, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the
E. coli ftsA
and
ftsZ
genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the
ftsA
and
ftsZ
genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.
High Rate of Uptake of Organic Nitrogen Compounds by
Prochlorococcus
Cyanobacteria as a Key to Their Dominance in Oligotrophic Oceanic Waters
Authors: Zubkov Mikhail V. , Fuchs Bernhard M. , Tarran Glen A. , Burkill Peter H. , Amann Rudolf
Published: February 2003

Direct evidence that marine cyanobacteria take up organic nitrogen compounds in situ at high rates is reported. About 33% of the total bacterioplankton turnover of amino acids, determined with a representative [
35
S]methionine precursor and flow sorting, can be assigned to
Prochlorococcus
spp. and 3% can be assigned to
Synechococcus
spp. in the oligotrophic and mesotrophic parts of the Arabian Sea, respectively. This finding may provide a mechanism for
Prochlorococcus
' competitive dominance over both strictly autotrophic algae and other bacteria in oligotrophic regions sustained by nutrient remineralization via a microbial loop.
Studies on the Mode of Action of Reutericyclin
Authors: Gänzle Michael G. , Vogel Rudi F.
Published: February 2003

The mode of action of reutericyclin was determined with fluorescent dyes that probed the permeability of the cytoplasmic membrane by large molecules, protons, and potassium. A comparison of reutericyclin activity with those of nisin, nigericin, and valinomycin demonstrated that reutericyclin does not form pores but selectively dissipates the transmembrane proton potential.
Cloning and Expression Analysis of the
pcbAB-pcbC
β-Lactam Genes in the Marine Fungus
Kallichroma tethys
Authors: Kim Chi-fai , Lee Simon K. Y. , Price Jackie , Jack Ralph W. , Turner Geoffrey , Kong Richard Y. C.
Published: February 2003

Here we report the identification of the β-lactam biosynthesis genes
pcbAB
and
pcbC
from a cosmid genomic DNA library of the marine fungus
Kallichroma tethys.
A BLAST homology search showed that they share high sequence identity with the δ-(
l
-α-aminoadipyl)-
l
-cysteinyl-
d
-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial β-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete
Acremonium chrysogenum
. Expression analysis by reverse transciption-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of
K. tethys
cultures. Complementation of an
Aspergillus nidulans
strain deficient in ACV synthetase suggested that at least
pcbAB
is functional, although attempts to isolate active antibiotic from
K. tethys
were unsuccessful.
Dibenzyl Sulfide Metabolism by White Rot Fungi
Authors: Van Hamme Jonathan D. , Wong Eddie T. , Dettman Heather , Gray Murray R. , Pickard Michael A.
Published: February 2003

Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization. Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied.
Trametes trogii
UAMH 8156,
Trametes hirsuta
UAMH 8165,
Phanerochaete chrysosporium
ATCC 24725,
Trametes versicolor
IFO 30340 (formerly
Coriolus
sp.), and
Tyromyces palustris
IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone. The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation. Laccase activity (0.15 U/ml) was detected in the
Trametes
cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence. These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.
Chlorine Inactivation of Spores of
Encephalitozoon
spp
Authors: Johnson C. H. , Marshall M. M. , DeMaria L. A. , Moffet J. M. , Korich D. G.
Published: February 2003

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of
Encephalitozoon
intestinalis
and to investigate the effect of chlorine on two other species,
E
cuniculi
and
E
.
hellem
, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.
UV Resistance of
Bacillus anthracis
Spores Revisited: Validation of
Bacillus subtilis
Spores as UV Surrogates for Spores of
B. anthracis
Sterne
Authors: Nicholson Wayne L. , Galeano Belinda
Published: February 2003

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of
Bacillus anthracis.
A review of the literature revealed that
B. anthracis
Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of
Bacillus subtilis
. To test this notion,
B. anthracis
Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of
B. subtilis
, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against
B. subtilis
spores are likely also sufficient to inactivate
B. anthracis
spores and that the spores of standard
B. subtilis
strains could reliably be used as a biodosimetry model for the UV inactivation of
B. anthracis
spores.
Recombinant Strain of
Bacillus thuringiensis
Producing Cyt1A, Cry11B, and the
Bacillus sphaericus
Binary Toxin
Authors: Park Hyun-Woo , Bideshi Dennis K. , Federici Brian A.
Published: February 2003

A novel recombinant
Bacillus thuringiensis
subsp.
israelensis
strain that produces the
B. sphaericus
binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC
50
] = 1.7 ng/ml) against fourth-instar
Culex quinquefasciatus
was higher than that of
B. thuringiensis
subsp.
israelensis
IPS-82 (LC
50
= 7.9 ng/ml) or
B. sphaericus
2362 (LC
50
= 12.6 ng/ml).
Molecular Analysis of a
Saccharomyces cerevisiae
Mutant with Improved Ability To Utilize Xylose Shows Enhanced Expression of Proteins Involved in Transport, Initial Xylose Metabolism, and the Pentose Phosphate Pathway
Authors: Wahlbom C. Fredrik , Cordero Otero Ricardo R. , van Zyl Willem H. , Hahn-Hägerdal Bärbel , Jönsson Leif J.
Published: February 2003

Differences between the recombinant xylose-utilizing
Saccharomyces cerevisiae
strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of
S. cerevisiae
TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of
S. cerevisiae
TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h
−1
, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter
−1
, 10 g of glucose plus 10 g of xylose liter
−1
or, for
S. cerevisiae
TMB 3400, 20 g of xylose liter
−1
.
S. cerevisiae
TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than
S. cerevisiae
TMB 3399. The biomass yields for
S. cerevisiae
TMB 3400 were 0.46 g of biomass g of consumed carbohydrate
−1
on glucose and 0.43 g of biomass g of consumed carbohydrate
−1
on xylose. A

K
s

value of 33 mM for xylose was obtained for
S. cerevisiae
TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in
S. cerevisiae
TMB 3400 than in
S. cerevisiae
TMB 3399 for (i)
HXT5
, encoding a hexose transporter; (ii)
XKS1
, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii)
SOL3
,
GND1
,
TAL1
, and
TKL1
, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of
XKS1
in
S. cerevisiae
TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for
S. cerevisiae
TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose.
Anaerobic Degradation of Ethylbenzene by a New Type of Marine Sulfate-Reducing Bacterium
Authors: Kniemeyer Olaf , Fischer Thomas , Wilkes Heinz , Glöckner Frank Oliver , Widdel Friedrich
Published: February 2003

Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene andm-xylene, respectively. However, strain EbS7 did not oxidize naphthalene,m-xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate,n-hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobicn-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.
The Abundance of Microcystin-Producing Genotypes Correlates Positively with Colony Size inMicrocystissp. and Determines Its Microcystin Net Production in Lake Wannsee
Authors: Kurmayer Rainer , Christiansen Guntram , Chorus Ingrid
Published: February 2003

The working hypotheses tested on a natural population ofMicrocystissp. in Lake Wannsee (Berlin, Germany) were that (i) the varying abundance of microcystin-producing genotypes versus non-microcystin-producing genotypes is a key factor for microcystin net production and (ii) the occurrence of a gene for microcystin net production is related to colony morphology, particularly colony size. To test these hypotheses, samples were fractionated by colony size with a sieving procedure during the summer of 2000. Each colony size class was analyzed for cell numbers, the proportion of microcystin-producing genotypes, and microcystin concentrations. The smallest size class ofMicrocystiscolonies (<50 μm) showed the lowest proportion of microcystin-producing genotypes, the highest proportion of non-microcystin-producing cells, and the lowest microcystin cell quotas (sum of microcystins RR, YR, LR, and WR). In contrast, the larger size classes ofMicrocystiscolonies (>100 μm) showed the highest proportion of microcystin-producing genotypes, the lowest proportion of non-microcystin-producing cells, and the highest microcystin cell quotas. The microcystin net production rate was nearly one to one positively related to the population growth rate for the larger colony size classes (>100 μm); however, no relationship could be found for the smaller size classes. It was concluded that the variations found in microcystin net production between colony size classes are chiefly due to differences in genotype composition and that the microcystin net production in the lake is mainly influenced by the abundance of the larger (>100-μm) microcystin-producing colonies.
A Sensitive and Inexpensive Yeast Bioassay for the Mycotoxin Zearalenone and Other Compounds with Estrogenic Activity
Authors: Mitterbauer Rudolf , Weindorfer Hanna , Safaie Naser , Krska Rudolf , Lemmens Marc , Ruckenbauer Peter , Kuchler Karl , Adam Gerhard
Published: February 2003

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of
Fusarium
. As a consequence of infection with
Fusarium culmorum
and
Fusarium graminearum
, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator
Saccharomyces cerevisiae
strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 μg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes
FUR1
and
URK1
, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic
Fusarium
mycotxins.
Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast
Pichia ciferrii
Authors: Bae Jung-Hoon , Sohn Jung-Hoon , Park Chang-Seo , Rhee Joon-Shick , Choi Eui-Sung
Published: February 2003

We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast
Pichia ciferrii
. The system uses (i) a mutagenized ribosomal protein L41 gene of
P. ciferrii
as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of
P. ciferrii
as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg of DNA was observed. To improve the de novo synthesis of sphingolipid, the
LCB2
gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from
P. ciferrii
and overexpressed under the control of the
P. ciferrii
glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an
LCB2
gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in
LCB2
mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.
Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments
Authors: Stamper David M. , Walch Marianne , Jacobs Rachel N.
Published: February 2003

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD
5
), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (
Escherichia coli
numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time.