bluuebunny/crossref_metadata_2025 huggingface dataset:
Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of
Listeria monocytogenes
Isolates
Authors: Ward Todd J. , Ducey Thomas F. , Usgaard Thomas , Dunn Katherine A. , Bielawski Joseph P.
Published: October 2008

Listeria monocytogenes
is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of
L. monocytogenes
lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of
L. monocytogenes
. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of
L. monocytogenes
. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in
inlA
associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.
Modulation of Chaperone Gene Expression in Mutagenized
Saccharomyces cerevisiae
Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins
Authors: Payne T. , Finnis C. , Evans L. R. , Mead D. J. , Avery S. V. , Archer D. B. , Sleep D.
Published: October 2008

The yeast
Saccharomyces cerevisiae
has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from
S. cerevisiae
, focusing predominantly on the products of the protein disulfide isomerase gene
PDI1
and the hsp70 gene
KAR2
. Here we show that the expression of the genes
SIL1, LHS1, JEM1
, and
SCJ1
, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.
Specific Binding of
Bacillus thuringiensis
Cry2A Insecticidal Proteins to a Common Site in the Midgut of
Helicoverpa
Species
Authors: Hernández-Rodríguez Carmen Sara , Van Vliet Adri , Bautsoens Nadine , Van Rie Jeroen , Ferré Juan
Published: October 2008

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with
125
I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of
Helicoverpa armigera
. Homologous-competition assays with
125
I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in
H. armigera
and
Helicoverpa zea
midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of
K

d

(dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding
R

t

(concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.
Bacteria Associated with Benthic Diatoms from Lake Constance: Phylogeny and Influences on Diatom Growth and Secretion of Extracellular Polymeric Substances
Authors: Bruckner Christian G. , Bahulikar Rahul , Rahalkar Monali , Schink Bernhard , Kroth Peter G.
Published: October 2008

The composition of diatom-associated bacterial communities was studied with 14 different unialgal xenic diatom cultures isolated from freshwater epilithic biofilms of Lake Constance, Germany. A clear dominance of
Alphaproteobacteria
was observed, followed by
Betaproteobacteria, Gammaproteobacteria, Bacteroidetes
, and
Verrucomicrobia
. Pure cultures of the diatom
Cymbella microcephala
, which was found to be dominant in epilithic biofilms in Lake Constance, were cocultivated with six associated bacterial strains. All these bacterial strains were able to grow in
C. microcephala
cultures in the absence of organic cosubstrates. Diatom growth was generally enhanced in the presence of bacteria, and polysaccharide secretion was generally increased in the presence of
Proteobacteria
. The monomer composition of extracellular polysaccharides of
C. microcephala
changed in relation to the presence of different bacteria, but the dominant monomers were less affected. Our results indicate that these changes were caused by the diatom itself rather than by specific bacterial degradation. One
Bacteroidetes
strain strongly influenced carbohydrate secretion by the alga via extracellular soluble compounds. Biofilms were formed only in the presence of bacteria. Phylogenetic analysis and coculture studies indicate an adaptation of
Proteobacteria
and
Bacteroidetes
to the microenvironment created by the diatom biofilm.
The Twin-Arginine Signal Peptide of
Bacillus subtilis
YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the
B. subtilis
Tat Pathway
Authors: Kolkman Marc A. B. , van der Ploeg René , Bertels Michael , van Dijk Maurits , van der Laan Joop , van Dijl Jan Maarten , Ferrari Eugenio
Published: October 2008

Proteins that are produced for commercial purposes in
Bacillus subtilis
are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in
B. subtilis
. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known
B. subtilis
Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the
B. subtilis
TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the
Bacillus licheniformis
α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an
Escherichia coli
Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in
B. subtilis
, as is the case with
E. coli
. We conclude that future applications for the Tat system of
B. subtilis
will most likely involve commercially interesting proteins that are Sec incompatible.
Identification of Components of Electron Transport Chains in the Extremely Thermoacidophilic Crenarchaeon
Metallosphaera sedula
through Iron and Sulfur Compound Oxidation Transcriptomes
Authors: Auernik Kathryne S. , Kelly Robert M.
Published: October 2008

The crenarchaeal order
Sulfolobales
collectively contain at least five major terminal oxidase complexes. Based on genome sequence information, all five complexes are found only in
Metallosphaera sedula
and
Sulfolobus tokodaii
, the two sequenced
Sulfolobales
capable of iron oxidization. While specific respiratory complexes in certain
Sulfolobales
have been characterized previously as proton pumps for maintaining intracellular pH and generating proton motive force, their contribution to sulfur and iron biooxidation has not been considered. For
M. sedula
growing in the presence of ferrous iron and reduced inorganic sulfur compounds (RISCs), global transcriptional analysis was used to track the response of specific genes associated with these complexes, as well as other known and putative respiratory electron transport chain elements. Open reading frames from all five terminal oxidase or
bc
1
-like complexes were stimulated on one or more conditions tested. Components of the
fox
(Msed0467 to Msed0489) and
soxNL
-
cbsABA
(Msed0500 to Msed0505) terminal/quinol oxidase clusters were triggered by ferrous iron, while the
soxABCDD
′ terminal oxidase cluster (Msed0285 to Msed0291) were induced by tetrathionate and S
0
. Chemolithotrophic electron transport elements, including a putative tetrathionate hydrolase (Msed0804), a novel polysulfide/sulfur/dimethyl sulfoxide reductase-like complex (Msed0812 to Msed0818), and a novel heterodisulfide reductase-like complex (Msed1542 to Msed1550), were also stimulated by RISCs. Furthermore, several hypothetical proteins were found to have strong responses to ferrous iron or RISCs, suggesting additional candidates in iron or sulfur oxidation-related pathways. From this analysis, a comprehensive model for electron transport in
M. sedula
could be proposed as the basis for examining specific details of iron and sulfur oxidation in this bioleaching archaeon.
Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia
Authors: Tracy Bryan P. , Gaida Stefan M. , Papoutsakis Eleftherios T.
Published: October 2008

The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe
Clostridium acetobutylicum
. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.
High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast
Kluyveromyces marxianus
DMKU3-1042
Authors: Nonklang Sanom , Abdel-Banat Babiker M. A. , Cha-aim Kamonchai , Moonjai Nareerat , Hoshida Hisashi , Limtong Savitree , Yamada Mamoru , Akada Rinji
Published: October 2008

We demonstrate herein the ability of
Kluyveromyces marxianus
to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to
Saccharomyces cerevisiae
. Growth and ethanol production by strains of
K. marxianus
and
S. cerevisiae
were compared under the same conditions.
K. marxianus
DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not
S. cerevisiae
, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a
K. marxianus
DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the
S. cerevisiae
Sc
URA3
gene. Surprisingly, Ura
+
transformants were easily obtained. By Southern blot hybridization, the linear Sc
URA3
DNA was found to have inserted randomly into the
K. marxianus
genome. Sequencing of one Lys
−
transformant confirmed the disruption of the Km
LYS1
gene by the Sc
URA3
insertion. A PCR-amplified linear DNA lacking
K. marxianus
sequences but containing an
Aspergillus
α-amylase gene under the control of the Sc
TDH3
promoter together with an Sc
URA3
marker was subsequently used to transform
K. marxianus
DMKU3-1042 in order to obtain transformants expressing
Aspergillus
α-amylase. Our results demonstrate that
K. marxianus
DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of
S. cerevisiae
-based molecular genetic tools.
4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of
Mucor mucedo
: Purification, Cloning of the Corresponding Gene, and Developmental Expression
Authors: Wetzel Jana , Scheibner Olaf , Burmester Anke , Schimek Christine , Wöstemeyer Johannes
Published: October 2008

The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of
Mucor mucedo
was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene,
TSP2
, was isolated. A labeled
TSP2
hybridization probe detects a single-copy gene in the genome of
M. mucedo
. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of
M. mucedo
. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the
TSP2
gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the
TSP2
genes in the three sequenced zygomycetous genomes (
Phycomyces blakesleeanus
,
Rhizopus oryzae
, and
Mucor circinelloides
), the closest relative is the
Myxococcus xanthus CsgA
gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.
Copy Number Suppressors of the
Aspergillus nidulans nimA1
Mitotic Kinase Display Distinctive and Highly Dynamic Cell Cycle-Regulated Locations
Authors: Ukil Leena , Varadaraj Archana , Govindaraghavan Meera , Liu Hui-Lin , Osmani Stephen A.
Published: October 2008

The
Aspergillus nidulans
NIMA kinase is essential for mitosis and is the founding member of the conserved
N
IMA-r
e
lated
k
inase (Nek) family of protein kinases. To gain insight into NIMA function, a copy number suppression screen has been completed that defines three proteins termed MCNA, MCNB, and MCNC (multi-copy-number suppressor of
nimA1
A, B, and C). All display a distinctive and dynamic cell cycle-specific distribution. MCNC has weak similarity to
Saccharomyces cerevisiae
Def1 within a shared CUE-like domain. MCNC, like Def1, is a cytoplasmic protein with slow mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its deletion causes polarization defects and a small colony phenotype. MCNC enters nuclei during mitosis. In contrast, MCNB is a nuclear protein displaying increased nuclear levels as cells progress through interphase but is lost from nuclei at mitosis. MCNB is highly related to the
Schizosaccharomyces pombe
forkhead transcription factor Sep1 and is likely a transcriptional activator of
nimA
. Most surprisingly, MCNA, a protein restricted to the aspergilli and pathogenic systemic dimorphic fungi (the
Eurotiomycetes
), defines a nuclear body located near nucleoli at the nuclear periphery of G
2
nuclei. During progression through mitosis, the MCNA body is excluded from nuclei. Cytoplasmic MCNA bodies then diminish during early stages of interphase, and single MCNA bodies are formed within nuclei as interphase progresses. Three sites of MCNA phosphorylation were mapped and mutated to implicate proline-directed phosphorylation in the equal segregation of MCNA during the cell cycle. The data indicate all three MCN proteins likely have cell cycle functions.
Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to
Ruminococcus albus
Authors: Ezer Anat , Matalon Erez , Jindou Sadanari , Borovok Ilya , Atamna Nof , Yu Zhongtang , Morrison Mark , Bayer Edward A. , Lamed Raphael
Published: October 2008

The rumen bacterium
Ruminococcus albus
binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.
Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes inStreptomyces clavuligerus
Authors: Zelyas Nathan J. , Cai Hui , Kwong Thomas , Jensen Susan E.
Published: October 2008

Streptomyces clavuligerusproduces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes inS. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genesorfA,orfB,orfC, ororfDwere unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid.orfAencodes a predicted hydroxymethyltransferase,orfBencodes a YjgF/YER057c/UK114-family regulatory protein,orfCencodes an aminotransferase, andorfDencodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants inorfCororfDalso accumulated a novel clavam metabolite instead of alanylclavam, and a complementedorfCmutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway.
Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in
Corynebacterium glutamicum
R
Authors: Tsuge Yota , Ogino Hidetaka , Teramoto Haruhiko , Inui Masayuki , Yukawa Hideaki
Published: October 2008

In previous work, random genome deletion mutants of
Corynebacterium glutamicum
R were generated using the insertion sequence (IS) element IS
31831
and the Cre/
loxP
excision system. One of these mutants,
C. glutamicum
strain RD41, resulting from the deletion of a 10.1-kb genomic region (ΔcgR_1595 through cgR_1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_1596 in WT
C. glutamicum
R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In
C. glutamicum
R, CgR_0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in
C. glutamicum
.
A Multicopper Oxidase (Cj1516) and a CopA Homologue (Cj1161) Are Major Components of the Copper Homeostasis System of
Campylobacter jejuni
Authors: Hall Stephen J. , Hitchcock Andrew , Butler Clive S. , Kelly David J.
Published: October 2008

Metal ion homeostasis mechanisms in the food-borne human pathogen
Campylobacter jejuni
are poorly understood. The Cj1516 gene product is homologous to the multicopper oxidase CueO, which is known to contribute to copper tolerance in
Escherichia coli
. Here we show, by optical absorbance and electron paramagnetic resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and trinuclear copper centers, which are characteristic of multicopper oxidases. Inductively coupled plasma mass spectrometry revealed that the protein contained approximately six copper atoms per polypeptide. The presence of an N-terminal “twin arginine” signal sequence suggested a periplasmic location for Cj1516, which was confirmed by the presence of
p
-phenylenediamine (
p
-PD) oxidase activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. Kinetic studies showed that the pure protein exhibited
p
-PD, ferroxidase, and cuprous oxidase activities and was able to oxidize an analogue of the bacterial siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake impairment was observed in a Cj1516 mutant. However, this mutant was very sensitive to increased copper levels in minimal media, suggesting a role in copper tolerance. This was supported by increased expression of the Cj1516 gene in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a putative CopA homologue, was also found to result in copper hypersensitivity, and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than either single mutant. These observations and the apparent lack of alternative copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major proteins involved in copper homeostasis in
C. jejuni
.
Regulation of Hemin Binding Proteins by a Novel Transcriptional Activator in
Porphyromonas gingivalis
Authors: Wu Jie , Lin Xinghua , Xie Hua
Published: October 2008

One of the features of the periodontal pathogen
Porphyromonas gingivalis
is the presence of complex iron acquisition systems that include an
hmuYRSTUV
locus. HmuY and HmuR are hemin binding proteins required for
P. gingivalis
growth. Previous studies have demonstrated that expression of the
hmu
locus is regulated in response to environmental changes, such as growth phases. However, the mechanisms involved in
hmu
gene regulation are poorly understood. Here we report that a novel transcriptional activator, PG1237, is required for the expression of
humY
and
humR
, but not other iron acquisition-related genes, such as
fetB
and
tlr
, which also encode hemin binding proteins. Real-time reverse transcription-PCR analysis revealed that a mutation in the pg1237 gene decreased expression of
hmuY
and
hmuR
149- and 25-fold, respectively, compared to that observed in the wild-type strain. In addition, differential expression of
hmuY
,
hmuR
, and the pg1237 gene was found to be quorum-sensing dependent, such that higher expression levels of these genes were observed when
P. gingivalis
was grown at a lower cell density, such as that seen during the early exponential growth phase. This work demonstrates the involvement of a novel transcriptional activator, PG1237, in expression of the
hmu
operon in a cell density-dependent fashion.
Posttranscriptional Regulation of Flagellin Synthesis in
Helicobacter pylori
by the RpoN Chaperone HP0958
Authors: Douillard Francois P. , Ryan Kieran A. , Caly Delphine L. , Hinds Jason , Witney Adam A. , Husain Sarah E. , O'Toole Paul W.
Published: October 2008

The
Helicobacter pylori
protein HP0958 is essential for flagellum biogenesis. It has been shown that HP0958 stabilizes the σ
54
factor RpoN. The aim of this study was to further investigate the role of HP0958 in flagellum production in
H. pylori
. Global transcript analysis identified a number of flagellar genes that were differentially expressed in an HP0958 mutant strain. Among these, the transcription of the major flagellin gene
flaA
was upregulated twofold, suggesting that HP0958 was a negative regulator of the
flaA
gene. However, the production of the FlaA protein was significantly reduced in the HP0958 mutant, and this was not due to the decreased stability of the FlaA protein. RNA stability analysis and binding assays indicated that HP0958 binds and destabilizes
flaA
mRNA. The HP0958 mutant was successfully complemented, confirming that the mutant phenotype described was due to the lack of HP0958. We conclude that HP0958 is a posttranscriptional regulator that modulates the amount of the
flaA
message available for translation in
H. pylori
.
Regulation of Fructose Transport and Its Effect on Fructose Toxicity in
Anabaena
spp
Authors: Ungerer Justin L. , Pratte Brenda S. , Thiel Teresa
Published: October 2008

Anabaena variabilis
grows heterotrophically using fructose, while the close relative
Anabaena
sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC transporter, herein named
frtRABC
, into
Anabaena
sp. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes are necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR,
frtA
was very highly expressed, with or without fructose. In the
frtR
mutant, fructose uptake was immediate, in contrast to that in the wild-type strain, which required about 40 min for induction of transport. In the
frtR
mutant, high-level expression of the fructose transporter resulted in cells that were extremely sensitive to fructose. Even in the presence of the inducer, fructose, expression of
frtA
was low in the wild-type strain compared to that in the
frtR
mutant, indicating that FrtR repressed the transporter genes even in the presence of fructose. FrtR bound to the upstream region of
frtA
, but binding was not visibly altered by fructose, further supporting the hypothesis that fructose has only a modest effect in relieving repression of
frtA
by FrtR.
A. variabilis
grew better with increasing concentrations of fructose up to 50 mM, showing increased cell size and heterocyst frequency.
Anabaena
sp. strain PCC 7120 did not show any of these changes when it was grown with fructose. Thus, although
Anabaena
sp. strain PCC 7120 could take up fructose and use it in the dark, fructose did not improve growth in the light.
Altered Oligomerization Properties of N316 Mutants ofEscherichia coliTyrR
Authors: Koyanagi Takashi , Katayama Takane , Suzuki Hideyuki , Kumagai Hidehiko
Published: October 2008

The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor ofEscherichia coliTyrRE274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrRN316Dand TyrRN316Rmutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrRN316Dand TyrRN316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrRN316D.
Induction of the Galactose Enzymes in
Escherichia coli
Is Independent of the C-1-Hydroxyl Optical Configuration of the Inducer
d
-Galactose
Authors: Lee Sang Jun , Lewis Dale E. A. , Adhya Sankar
Published: October 2008

The two optical forms of aldohexose galactose differing at the C-1 position, α-
d
-galactose and β-
d
-galactose, are widespread in nature. The two anomers also occur in di- and polysaccharides, as well as in glycoconjugates. The anomeric form of
d
-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other
d
-galactose-metabolizing enzymes are coded by genes that constitute an operon in
Escherichia coli
. The operon is repressed by the repressor GalR and induced by
d
-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of
d
-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the
gal
operon, specifically the mutarotase, be achievable by either anomer of
d
-galactose. Here we report in vivo and in vitro experiments showing that both α-
d
-galactose and β-
d
-galactose are capable of inducing transcription of the
gal
operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the α configuration inactivate the induction capacity of the sugar, the effect of substitutions in the β configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.
Identification of
Bdellovibrio bacteriovorus
HD100 Bd0714 as a Nudix dGTPase
Authors: Steyert Susan R. , Messing Simon A. J. , Amzel L. Mario , Gabelli Sandra B. , Piñeiro Silvia A.
Published: October 2008

Bdellovibrio bacteriovorus
bacteria are predatory organisms that attack other gram-negative bacteria. Here, we report that Bd0714 is a Nudix dGTPase from
B. bacteriovoru
s HD100 with a substrate specificity similar to that of
Escherichia coli
MutT and complements an
E. coli mutT
-deficient strain. We observed different transcription levels of the gene throughout the predator life cycle.
Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic
Synechococcus
sp. from Microbial Mats
Authors: Adams Melissa M. , Gómez-García María R. , Grossman Arthur R. , Bhaya Devaki
Published: October 2008

The genomes of two closely related thermophilic cyanobacterial isolates, designated
Synechococcus
isolate OS-A and
Synechococcus
isolate OS-B′, from the microbial mats of Octopus Spring (Yellowstone National Park) have been sequenced. An extensive suite of genes that are controlled by phosphate levels constitute the putative Pho regulon in these cyanobacteria. We examined physiological responses of an axenic OS-B′ isolate as well as transcript abundances of Pho regulon genes as the cells acclimated to phosphorus-limiting conditions. Upon imposition of phosphorus deprivation, OS-B′ stopped dividing after three to four doublings, and absorbance spectra measurements indicated that the cells had lost most of their phycobiliproteins and chlorophyll
a
. Alkaline phosphatase activity peaked and remained high after 48 h of phosphorus starvation, and there was an accumulation of transcripts from putative Pho regulon genes. Interestingly, the genome of
Synechococcus
isolate OS-B′ harbors a cluster of
phn
genes that are not present in OS-A isolates. The proteins encoded by the
phn
genes function in the transport and metabolism of phosphonates, which could serve as an alternative phosphorus source when exogenous phosphate is low. The
phn
genes were upregulated within a day of eliminating the source of phosphate from the medium. However, the ability of OS-B′ to utilize methylphosphonate as a sole phosphorus source occurred only after an extensive period of exposure to the substrate. Once acclimated, the cells grew rapidly in fresh medium with methylphosphonate as the only source of phosphorus. The possible implications of these results are discussed with respect to the ecophysiology of the microbial mats.
Sequence and Analysis of a Plasmid-Encoded Mercury Resistance Operon from
Mycobacterium marinum
Identifies MerH, a New Mercuric Ion Transporter
Authors: Schué Mathieu , Dover Lynn G. , Besra Gurdyal. S. , Parkhill Julian , Brown Nigel L.
Published: October 2008

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg
2+
transporter. MerH was found to transport mercuric ions in
Escherichia coli
via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.
Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of
Brucella abortus
Authors: Bukata Lucas , Altabe Silvia , de Mendoza Diego , Ugalde Rodolfo A. , Comerci Diego J.
Published: October 2008

The
Brucella
cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for
Brucella
virulence. In this study we investigated the first step of PE biosynthesis in
B. abortus
, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of
pssA
abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for
Brucella
viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of
B. abortus
, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative
Brucella
-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of
B. abortus
with the host cell.
Amino Acid Substitutions in Transmembrane Domains 9 and 10 of GerVB That Affect the Germination Properties of
Bacillus megaterium
Spores
Authors: Christie Graham , Lowe Christopher R.
Published: October 2008

The molecular basis for differences in germinant recognition of
Bacillus megaterium
QM B1551 spores containing the GerVB and/or GerUB receptor proteins has been examined by site-directed mutagenesis and the construction of cross-homologue chimeras. Focusing on nonconserved residues predicted to reside in transmembrane domains 9 and 10, we demonstrate that GerVB residues Ser319 and Leu345 are of particular importance in defining the specificity and apparent affinity of the receptor for germinants. Kinetic analyses of mutants with different amino acid substitutions at these positions indicate that Ser319 and Leu345 are not involved directly in the binding of germinants, but probably reside in regions of the receptor where structural perturbations can affect the conformation of, or access to, germinant binding sites. Position 345 is also shown to be of importance in GerUB, where the F345A mutation severely impairs receptor function. Functionality is restored in the GerUB Ala345 background by substituting putative outer-loop residues adjacent to TM10 for the corresponding residues in GerVB, indicating that a degree of structural coordination between these regions is important to receptor function.
Macroamphiphilic Components of Thermophilic Actinomycetes: Identification of Lipoteichoic Acid in
Thermobifida fusca
Authors: Rahman Obaidur , Pfitzenmaier Markus , Pester Oxana , Morath Siegfried , Cummings Stephen P. , Hartung Thomas , Sutcliffe Iain C.
Published: October 2008

The cell envelopes of gram-positive bacteria contain structurally diverse membrane-anchored macroamphiphiles (lipoteichoic acids and lipoglycans) whose functions are poorly understood. Since regulation of membrane composition is an important feature of adaptation to life at higher temperatures, we have examined the nature of the macroamphiphiles present in the thermophilic actinomycetes
Thermobifida fusca
and
Rubrobacter xylanophilus
. Following hot-phenol-water extraction and purification by hydrophobic interaction chromatography, Western blotting with a monoclonal antibody against lipoteichoic acid strongly suggested the presence of a polyglycerophosphate lipoteichoic acid in
T. fusca
. This structure was confirmed by chemical and nuclear magnetic resonance analyses, which confirmed that the lipoteichoic acid is substituted with β-glucosyl residues, in common with the teichoic acid of this organism. In contrast, several extraction methods failed to recover significant macroamphiphilic carbohydrate- or phosphate-containing material from
R. xylanophilus
, suggesting that this actinomycete most likely lacks a membrane-anchored macroamphiphile. The finding of a polyglycerophosphate lipoteichoic acid in
T. fusca
suggests that lipoteichoic acids may be more widely present in the cell envelopes of actinomycetes than was previously assumed. However, the apparent absence of macroamphiphiles in the cell envelope of
R. xylanophilus
is highly unusual and suggests that macroamphiphiles may not always be essential for cell envelope homeostasis in gram-positive bacteria.
Proteasomal Components Required for Cell Growth and Stress Responses in the Haloarchaeon
Haloferax volcanii
Authors: Zhou Guangyin , Kowalczyk David , Humbard Matthew A. , Rohatgi Sunil , Maupin-Furlow Julie A.
Published: October 2008

Little is known regarding the biological roles of archaeal proteases. The haloarchaeon
Haloferax volcanii
is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of
H. volcanii
mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core α1 (
psmA
), β (
psmB
), and α2 (
psmC
) subunits as well as genes (
panA
and
panB
) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either α1 or α2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either β alone or α1 and α2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the
panA panB
double knockout strain. Deletion of genes encoding either α1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of
l
-canavanine. Abolishment of α1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for
panA
knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.
Loss of a Biofilm-Inhibiting Glycosyl Hydrolase during the Emergence of
Yersinia pestis
Authors: Erickson David L. , Jarrett Clayton O. , Callison Julie A. , Fischer Elizabeth R. , Hinnebusch B. Joseph
Published: October 2008

Yersinia pestis
, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related
Yersinia pseudotuberculosis
, from which
Y. pestis
recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut.
Y. pestis
biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the
hms
genes; identical genes are present in
Y. pseudotuberculosis
. The
Yersinia
Hms proteins contain functional domains present in
Escherichia coli
and
Staphylococcus
proteins known to synthesize a poly-β-1,6-
N
-acetyl-
d
-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of
Y. pestis
and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of
Y. pseudotuberculosis
that cleaved β-linked
N
-acetylglucosamine residues and reduced biofilm formation by staphylococci and
Y. pestis
in vitro. The
Y. pestis nghA
ortholog is a pseudogene, and overexpression of functional
nghA
reduced ECM surface accumulation and inhibited the ability of
Y. pestis
to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in
Y. pestis
may have contributed to the recent evolution of flea-borne transmission.
Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon
Haloferax volcanii
Authors: Plavner Noa , Eichler Jerry
Published: October 2008

In
Eukarya
, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For
Archaea
, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of
Haloferax volcanii
proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the
H. volcanii
S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the
H. volcanii
S-layer glycoprotein as occurring within the cell.
A Novel Class of Modular Transporters for Vitamins in Prokaryotes
Authors: Rodionov Dmitry A. , Hebbeln Peter , Eudes Aymerick , ter Beek Josy , Rodionova Irina A. , Erkens Guus B. , Slotboom Dirk J. , Gelfand Mikhail S. , Osterman Andrei L. , Hanson Andrew D. , Eitinger Thomas
Published: October 2008

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.
An Essential DnaB Helicase ofBacillus anthracis: Identification, Characterization, and Mechanism of Action
Authors: Biswas Esther E. , Barnes Marjorie H. , Moir Donald T. , Biswas Subhasis B.
Published: October 2008

We have described a novel essential replicative DNA helicase fromBacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaBBA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaBBAdisplayed distinct enzymatic and kinetic properties. DnaBBAhas low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaBBAwas coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaBBAdemonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaBBAhas an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaBBAis highly efficient in DNA unwinding and its coupling to ATP consumption.