bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu
Authors: Lamberg Arja , Nieminen Sari , Qiao Mingqiang , Savilahti Harri 
Published: February 2003

ABSTRACT

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10
4
to 10
6
CFU/μg of input transposon DNA in the four species tested, i.e.,
Escherichia coli
,
Salmonella enterica
serovar Typhimurium,
Erwinia carotovora
, and
Yersinia enterocolitica
. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.
Impact of Transport Crate Reuse and of Catching and Processing on
Campylobacter
and
Salmonella
Contamination of Broiler Chickens
Authors: Slader J. , Domingue G. , Jørgensen F. , McAlpine K. , Owen R. J. , Bolton F. J. , Humphrey T. J. 
Published: February 2003

ABSTRACT

The influence of transport, catching, and processing on contamination of broiler chickens with
Salmonella
and
Campylobacter
was investigated. Transport crates were reused with high frequency and were often still contaminated with
Salmonella
and
Campylobacter
when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously
Campylobacter
- and
Salmonella
-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The
Campylobacter
types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with
Campylobacter
(
P
< 0.001).
Identification, Characterization, and Expression of a Second, Bicistronic, Operon Involved in the Production of Lactocin S in
Lactobacillus sakei
L45
Authors: Skaugen Morten , Andersen Elizabeth L. , Christie Vigdis H. , Nes Ingolf F. 
Published: February 2003

ABSTRACT

Through the analysis of spontaneous insertion mutants of
Lactobacillus sakei
L45, a second operon involved in lactocin S production was identified and characterized. The new, bicistronic unit, termed
lasXY
, is situated immediately upstream of the previously characterized nine-open reading frame (ORF) lactocin S operon (
lasA-W
) and is transcribed in the opposite direction. The proximal of the two newly identified genes,
lasX
, specifies a 285-residue protein that is similar to a group of proteins with reported gene regulation functions in gram-positive bacteria. It was demonstrated that the
lasX
mutants have a strongly reduced level of
lasA
and
lasA-W
mRNA, thus indicating the likely cause of the Bac
−
phenotype of these mutants. The second ORF in the operon,
lasY
, specifies a 300-residue ABC transporter homolog, the function of which is currently obscure. Transcription initiation mapping of the
lasXY
operon demonstrates that the two lactocin S promoters overlap such that both transcripts initiate within the −35 region of the oppositely oriented promoter. This organization of promoters is unique among this group of regulons and may constitute a modulatory site in the proposed LasX-dependent expression of
lasA
and downstream genes.
Frequency of Bacteriocin Resistance Development and Associated Fitness Costs in
Listeria monocytogenes
Authors: Gravesen A. , Jydegaard Axelsen A.-M. , Mendes da Silva J. , Hansen T. B. , Knøchel S. 
Published: February 2003

ABSTRACT

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in
Listeria monocytogenes
to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10
−6
. However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10
−7
to 10
−2
. Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10°C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 × 10
−8
). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5°C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5°C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.
Genomic Diversity within the Genus
Pediococcus
as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis
Authors: Simpson P. J. , Stanton C. , Fitzgerald G. F. , Ross R. P. 
Published: February 2003

ABSTRACT

The genomic diversity of 33 previously assigned strains from six species within the genus
Pediococcus
was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes
Apa
I,
Not
I, and
Asc
I produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the
Not
I and
Asc
I macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with
Asc
I, all
Pediococcus parvulus
strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three
P. damnosus
and two
P. pentosaceus
strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.
Use of a Marker Organism To Model the Spread of Central Nervous System Tissue in Cattle and the Abattoir Environment during Commercial Stunning and Carcass Dressing
Authors: Daly D. J. , Prendergast D. M. , Sheridan J. J. , Blair I. S. , McDowell D. A. 
Published: February 2003

ABSTRACT

Due to concerns about a link between variant Creutzfeldt-Jakob disease in humans and similar prion protein-induced disease in cattle, i.e., bovine spongiform encephalopathy (BSE), strict controls are in place to exclude BSE-positive animals and/or specified risk materials including bovine central nervous system (CNS) tissue from the human food chain. However, current slaughter practice, using captive bolt guns, may induce disruption of brain tissues and mobilize CNS tissues into the bovine circulatory system, leading to the dispersion of CNS tissues (including prion proteins) throughout the derived carcass. This project used a marker (antibiotic-resistant) strain of
Pseudomonas fluorescens
to model the effects of commercial captive bolt stunning procedures on the movement of mobilized CNS material within slaughtered animals and the abattoir environment. The marker organism, introduced by injection through the bolt entry aperture or directly using a cartridge-fired captive bolt, was detected in the slaughter environment immediately after stunning and in the abattoir environment at each subsequent stage of the slaughter-dressing process. The marker organism was also detected on the hands of operatives; on slaughter equipment; and in samples of blood, organs, and musculature of inoculated animals. There were no significant differences between the results obtained by the two inoculation methods (
P
< 0.05). This study demonstrates that material present in, or introduced into, the CNS of cattle during commercial captive bolt stunning may become widely dispersed across the many animate and inanimate elements of the slaughter-dressing environment and within derived carcasses including meat entering the human food chain.
Optimization of Reverse Transcriptase PCR To Detect Viable Shiga-Toxin-Producing
Escherichia coli
Authors: McIngvale S. C. , Elhanafi D. , Drake M. A. 
Published: February 2003

ABSTRACT

The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing
Escherichia coli
(STEC) was investigated. Four primer sets, each targeting a specific region in the
slt-II
operon, were evaluated for their stringency and specificity for
slt-II
mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of
E. coli slt-II
mRNA is useful for detection of viable STEC in ground beef.
Identification of a Bacterium That Specifically Catalyzes the Reductive Dechlorination of Polychlorinated Biphenyls with Doubly Flanked Chlorines
Authors: Wu Qingzhong , Watts Joy E. M. , Sowers Kevin R. , May Harold D. 
Published: February 2003

ABSTRACT

A microorganism whose growth is linked to the dechlorination of polychlorinated biphenyls (PCBs) with doubly flanked chlorines was identified. Identification was made by reductive analysis of community 16S ribosomal DNA (rDNA) sequences from a culture enriched in the presence of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB), which was dechlorinated at the
para
position. Denaturing gradient gel electrophoresis (DGGE) analysis of total 16S rDNA extracted from the culture led to identification of three operational taxonomic units (OTUs 1, 2, and 3). OTU 1 was always detected when 2,3,4,5-CB or other congeners with doubly flanked chlorines were present and dechlorinated. Only OTUs 2 and 3 were detected in the absence of PCBs and when other PCBs (i.e., PCBs lacking doubly flanked chlorines) were not dechlorinated. Partial sequences of OTUs 2 and 3 exhibited 98.2% similarity to the sequence of “
Desulfovibrio caledoniensis
” (accession no.
DCU53465
). A sulfate-reducing vibrio isolated from the culture generated OTUs 2 and 3. This organism could not dechlorinate 2,3,4,5-CB. From these results we concluded that OTU 1 represents the dechlorinating bacterium growing in a coculture with a
Desulfovibrio
sp. The 16S rDNA sequence of OTU 1 is most similar to the 16S rDNA sequence of bacterium
o
-17 (89% similarity), an
ortho
-PCB-dechlorinating bacterium. The PCB dechlorinator, designated bacterium DF-1, reductively dechlorinates congeners with doubly flanked chlorines when it is supplied with formate or H
2
-CO
2
(80:20).
Characterization of Glycine Betaine Porter I from
Listeria monocytogenes
and Its Roles in Salt and Chill Tolerance
Authors: Mendum Mary Lou , Smith Linda Tombras 
Published: February 2003

ABSTRACT

Listeria monocytogenes
is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in
L. monocytogenes
is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na
+
-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and γ-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed
L. monocytogenes
grown in the presence of glycine betaine.
Biodiversity of the Bacterial Flora on the Surface of a Smear Cheese
Authors: Brennan Noelle M. , Ward Alan C. , Beresford Thomas P. , Fox Patrick F. , Goodfellow Michael , Cogan Timothy M. 
Published: February 2003

ABSTRACT

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with
Brevibacterium linens
BL2) and noninoculated (not deliberately inoculated with
B. linens
BL2) cheese were investigated. The results show that, contrary to accepted belief,
B. linens
is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative
Staphylococcus
spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of
Corynebacterium casei
(50.2% of isolates),
Corynebacterium mooreparkense
(26% of isolates), and
Microbacterium gubbeenense
(12.8% of isolates). In addition, five of the isolates from the inoculated cheese were
Corynebacterium flavescens
. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species.
C. mooreparkense
and
C. casei
grew at pH values below 4.9 in the presence of 8% NaCl, while
M. gubbeenense
did not grow below pH 5.8 in the presence of 5 to 10% NaCl.
B. linens
BL2 was not recovered from the inoculated cheese because it was inhibited by all the
Staphylococcus
isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.
Molecular Relationship between Two Groups of the Genus
Leptospirillum
and the Finding that
Leptospirillum ferriphilum
sp. nov. Dominates South African Commercial Biooxidation Tanks That Operate at 40°C
Authors: Coram Nicolette J. , Rawlings Douglas E. 
Published: February 2003

ABSTRACT

Iron-oxidizing bacteria belonging to the genus
Leptospirillum
are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40°C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of
rrn
genes, and based on 16S rRNA sequence data, these isolates clustered together with the
Leptospirillum ferrooxidans
type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of
rrn
genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name
Leptospirillum ferriphilum
sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (
L. ferrooxidans
), were capable of growth at 45°C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.
Calnexin Overexpression Increases Manganese Peroxidase Production in
Aspergillus niger
Authors: Conesa Ana , Jeenes David , Archer David B. , van den Hondel Cees A. M. J. J. , Punt Peter J. 
Published: February 2003

ABSTRACT

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the
Phanerochaete chrysosporium
manganese peroxidase (MnP) in
Aspergillus niger
resulted in an increase in the expression level of the
clxA
and
bipA
genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of
clxA
and
bipA
was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas
bipA
overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.
Identical Ring Cleavage Products during Anaerobic Degradation of Naphthalene, 2-Methylnaphthalene, and Tetralin Indicate a New Metabolic Pathway
Authors: Annweiler Eva , Michaelis Walter , Meckenstock Rainer U. 
Published: February 2003

ABSTRACT

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C
1
unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C
11
H
16
O
4
(C
11
H
16
O
4
-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by
β
-oxidation of one of the side chains of the C
11
H
16
O
4
-diacid. Stable isotope-labeling growth experiments with either
13
C-labeled naphthalene, per-deuterated naphthalene-
d
8
, or a
13
C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.