bluuebunny/crossref_metadata_2025_split huggingface dataset:
AguR Is Required for Induction of the
Streptococcus mutans
Agmatine Deiminase System by Low pH and Agmatine
Authors: Liu Yaling , Zeng Lin , Burne Robert A. 
Published: March 2009

ABSTRACT

Acidic conditions and the presence of exogenous agmatine are required to achieve maximal expression of the agmatine deiminase system (AgDS) of
Streptococcus mutans
. Here we demonstrate that the transcriptional activator of the AgDS, AguR, is required for the responses to agmatine and to low pH. Linker scanning mutagenesis was used to create a panel of mutated
aguR
genes that were utilized to complement an
aguR
deletion mutant of
S. mutans
. The level of production of the mutant proteins was shown to be comparable to that of the wild-type AguR protein. Mutations in the predicted DNA binding domain of AguR eliminated activation of the
agu
operon. Insertions into the region connecting the DNA binding domain to the predicted extracellular and transmembrane domains were well tolerated. In contrast, a variety of mutants were isolated that had a diminished capacity to respond to low pH but retained the ability to activate AgDS gene expression in response to agmatine, and vice versa. Also, a number of mutants were unable to respond to either agmatine or low pH. AguD, which is a predicted agmatine-putrescine antiporter, was found to be a negative regulator of AgDS gene expression in the absence of exogenous agmatine but was not required for low-pH induction of the AgDS genes. This study reveals that the control of AgDS gene expression by both agmatine and low pH is coordinated through the AguR protein and begins to identify domains of the protein involved in sensing and signaling.
UV Light Inactivation of Bacterial Biothreat Agents
Authors: Rose L. J. , O'Connell H. 
Published: March 2009

ABSTRACT

Seven species of bacterial biothreat agents were tested for susceptibility to UV light (254 nm). All gram-negative organisms tested required <12 mJ/cm
2
for a 4-log
10
reduction in viability (inactivation). Tailing off of the
B. anthracis
spore inactivation curves began close to the 2-log
10
inactivation point, with a fluence of approximately 40 mJ/cm
2
, and 3-log
10
inactivation still was not achieved with a fluence of 120 mJ/cm
2
.
Generation of Single-Copy Transposon Insertions in
Clostridium perfringens
by Electroporation of Phage Mu DNA Transposition Complexes
Authors: Lanckriet A. , Timbermont L. , Happonen L. J. , Pajunen M. I. , Pasmans F. , Haesebrouck F. , Ducatelle R. , Savilahti H. , Van Immerseel F. 
Published: March 2009

ABSTRACT

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in
Clostridium perfringens
has been restricted to the use of Tn
916
-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in
C. perfringens
, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of
C. perfringens
.
Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete
Ceriporiopsis subvermispora
Authors: Harreither Wolfgang , Sygmund Christoph , Dünhofen Evelyn , Vicuña Rafael , Haltrich Dietmar , Ludwig Roland 
Published: March 2009

ABSTRACT

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier
Ceriporiopsis subvermispora
when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one
cdh
gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of
C. subvermispora
CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.
Cloning and Characterization of the Biosynthetic Gene Cluster for Tomaymycin, an SJG-136 Monomeric Analog
Authors: Li Wei , Chou ShenChieh , Khullar Ankush , Gerratana Barbara 
Published: March 2009

ABSTRACT

Tomaymycin produced by
Streptomyces achromogenes
is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.
Biosynthesis of Sibiromycin, a Potent Antitumor Antibiotic
Authors: Li Wei , Khullar Ankush , Chou ShenChieh , Sacramo Ashley , Gerratana Barbara 
Published: March 2009

ABSTRACT
Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compounds with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clinical trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement experiments. The sibiromycin gene cluster consisting of 26 open reading frames reveals a “modular” strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addition, the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chemical complementation studies are reported to support the proposed biosynthetic pathway.
Lateral Transfer of Genes for Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Degradation
Authors: Andeer Peter F. , Stahl David A. , Bruce Neil C. , Strand Stuart E. 
Published: March 2009

ABSTRACT

Recent studies demonstrated that degradation of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by species of
Rhodococcus
,
Gordonia
, and
Williamsia
is mediated by a novel cytochrome P450 with a fused flavodoxin reductase domain (XplA) in conjunction with a flavodoxin reductase (XplB). Pulse field gel analysis was used to localize
xplA
to extrachromosomal elements in a
Rhodococcus
sp. and distantly related
Microbacterium
sp. strain MA1. Comparison of
Rhodococcus rhodochrous
11Y and
Microbacterium
plasmid sequences in the vicinity of
xplB
and
xplA
showed near identity (6,710 of 6,721 bp). Sequencing of the associated 52.2-kb region of the
Microbacterium
plasmid pMA1 revealed flanking insertion sequence elements and additional genes implicated in RDX uptake and degradation.
Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of
Cronobacter
Genomospecies (
Enterobacter sakazakii
) in Powdered Infant Formula
Authors: Almeida C. , Azevedo N. F. , Iversen C. , Fanning S. , Keevil C. W. , Vieira M. J. 
Published: March 2009

ABSTRACT

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of
Cronobacter
strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several
Enterobacteriaceae
species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of
Cronobacter
in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.
Transfer of Antibiotic Resistance Marker Genes between Lactic Acid Bacteria in Model Rumen and Plant Environments
Authors: Toomey Niamh , Monaghan Áine , Fanning Séamus , Bolton Declan 
Published: March 2009

ABSTRACT

Three wild-type dairy isolates of lactic acid bacteria (LAB) and one
Lactococcus lactis
control strain were analyzed for their ability to transfer antibiotic resistance determinants (plasmid or transposon located) to two LAB recipients using both in vitro methods and in vivo models. In vitro transfer experiments were carried out with the donors and recipients using the filter mating method. In vivo mating examined transfer in two natural environments, a rumen model and an alfalfa sprout model. All transconjugants were confirmed by Etest, PCR, pulsed-field gel electrophoresis, and Southern blotting. The in vitro filter mating method demonstrated high transfer frequencies between all LAB pairs, ranging from 1.8 × 10
−5
to 2.2 × 10
−2
transconjugants per recipient. Transconjugants were detected in the rumen model for all mating pairs tested; however, the frequencies of transfer were low and inconsistent over 48 h (ranging from 1.0 × 10
−9
to 8.0 × 10
−6
transconjugants per recipient). The plant model provided an environment that appeared to promote comparatively higher transfer frequencies between all LAB pairs tested over the 9-day period (transfer frequencies ranged from 4.7 × 10
−4
to 3.9 × 10
−1
transconjugants per recipient). In our test models, dairy cultures of LAB can act as a source of mobile genetic elements encoding antibiotic resistance that can spread to other LAB. This observation could have food safety and public health implications.
PCR-Based Method Using Propidium Monoazide To Distinguish Viable from Nonviable
Bacillus subtilis
Spores
Authors: Rawsthorne H. , Dock C. N. , Jaykus L. A. 
Published: March 2009

ABSTRACT

This paper describes a molecular-based method which is able to discriminate between viable and inactivated
Bacillus subtilis
spores by utilizing the DNA-intercalating dye propidium monoazide. The approach should be valuable in our attempt to employ molecular methods to streamline the evaluation of process validation using bacterial endospores.
Eukaryotic
nirK
Genes Encoding Copper-Containing Nitrite Reductase: Originating from the Protomitochondrion?
Authors: Kim Sang-Wan , Fushinobu Shinya , Zhou Shengmin , Wakagi Takayoshi , Shoun Hirofumi 
Published: March 2009

ABSTRACT

Although denitrification or nitrate respiration has been found among a few eukaryotes, its phylogenetic relationship with the bacterial system remains unclear because orthologous genes involved in the bacterial denitrification system were not identified in these eukaryotes. In this study, we isolated a gene from the denitrifying fungus
Fusarium oxysporum
that is homologous to the bacterial
nirK
gene responsible for encoding copper-containing nitrite reductase (NirK). Characterization of the gene and its recombinant protein showed that the fungal
nirK
gene is the first eukaryotic ortholog of the bacterial counterpart involved in denitrification. Additionally, recent genome analyses have revealed the occurrence of
nirK
homologs in many fungi and protozoa, although the denitrifying activity of these eukaryotes has never been examined. These eukaryotic homolog genes, together with the fungal
nirK
gene of
F
.
oxysporum
, are grouped in the same branch of the phylogenetic tree as the
nirK
genes of bacteria, archaea, and eukaryotes, implying that eukaryotic
nirK
and its homologs evolved from a single ancestor (possibly the protomitochondrion). These results show that the fungal denitrifying system has the same origin as its bacterial counterpart.
Comparative Genomic Hybridization Analysis of Two Predominant Nordic Group I (Proteolytic)Clostridium botulinumType B Clusters
Authors: Lindström Miia , Hinderink Katja , Somervuo Panu , Kiviniemi Katri , Nevas Mari , Chen Ying , Auvinen Petri , Carter Andrew T. , Mason David R. , Peck Michael W. , Korkeala Hannu 
Published: March 2009

ABSTRACTComparative genomic hybridization analysis of 32 Nordic group IClostridium botulinumtype B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group IC. botulinumtype B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.
Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic
Clostridium botulinum
Bacteria
Authors: Stringer Sandra C. , Webb Martin D. , Peck Michael W. 
Published: March 2009

ABSTRACT

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic
Clostridium botulinum
Eklund 17B. The times to germination (
t
germ
), one mature cell (
t
C1
), and two mature cells (
t
C2
) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the
t
germ
, the outgrowth time (
t
C1
−
t
germ
), and the first doubling time (
t
C2
−
t
C1
). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (
t
C1
) and outgrowth (
t
C1
−
t
germ
) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.
Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from
Marinobacter aquaeolei
VT8
Authors: Wahlen Bradley D. , Oswald Whitney S. , Seefeldt Lance C. , Barney Brett M. 
Published: March 2009

ABSTRACT

Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium
Marinobacter aquaeolei
VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in
Escherichia coli
as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP
+
. The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed.
Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas
Authors: Hodgetts Jennifer , Boonham Neil , Mumford Rick , Dickinson Matthew 
Published: March 2009

ABSTRACTPrimers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.
Striking Diversity of
vmp1
, a Variable Gene Encoding a Putative Membrane Protein of the Stolbur Phytoplasma
Authors: Cimerman Agnès , Pacifico Davide , Salar Pascal , Marzachì Cristina , Foissac Xavier 
Published: March 2009

ABSTRACT

Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the “
Candidatus
Phytoplasma asteris” genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The
vmp1
gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to
vmp1
in the genomes of nine StolP isolates.
vmp1
is variable in size, and eight different
vmp1
RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of
vmp1
sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates.
vmp1
appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.
Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice
Authors: Simon Carola , Herath Judith , Rockstroh Stephanie , Daniel Rolf 
Published: March 2009

ABSTRACT

Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an
Escherichia coli polA
mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.
Phylogenetic and Chemical Diversity of Three Chemotypes of Bloom-Forming
Lyngbya
Species (
Cyanobacteria
:
Oscillatoriales
) from Reefs of Southeastern Florida
Authors: Sharp Koty , Arthur Karen E. , Gu Liangcai , Ross Cliff , Harrison Genelle , Gunasekera Sarath P. , Meickle Theresa , Matthew Susan , Luesch Hendrik , Thacker Robert W. , Sherman David H. , Paul Valerie J. 
Published: March 2009

ABSTRACT

The cyanobacterial genus
Lyngbya
includes free-living, benthic, filamentous cyanobacteria that form periodic nuisance blooms in lagoons, reefs, and estuaries.
Lyngbya
spp. are prolific producers of biologically active compounds that deter grazers and help blooms persist in the marine environment. Here, our investigations reveal the presence of three distinct
Lyngbya
species on nearshore reefs in Broward County, FL, sampled in 2006 and 2007. With a combination of morphological measurements, molecular biology techniques, and natural products chemistry, we associated these three
Lyngbya
species with three distinct
Lyngbya
chemotypes. One species, identified as
Lyngbya
cf.
confervoides
via morphological measurements and 16S rRNA gene sequencing, produces a diverse array of bioactive peptides and depsipeptides. Our results indicate that the other two
Lyngbya
species produce either microcolins A and B or curacin D and dragonamides C and D. Results from screening for the biosynthetic capacity for curacin production among the three
Lyngbya
chemotypes in this study correlated that capacity with the presence of curacin D. Our work on these bloom-forming
Lyngbya
species emphasizes the significant phylogenetic and chemical diversity of the marine cyanobacteria on southern Florida reefs and identifies some of the genetic components of those differences.