bluuebunny/crossref_metadata_2025 huggingface dataset:
Does the Trigger for Abrupt Climate Change Reside in the Ocean or in the Atmosphere?
Authors: Broecker W. S.
Published: June 2003

Two hypotheses have been put forward to explain the large and abrupt climate changes that punctuated glacial time. One attributes such changes to reorganizations of the ocean's thermohaline circulation and the other to changes in tropical atmosphere-ocean dynamics. In an attempt to distinguish between these hypotheses, two lines of evidence are examined. The first involves the timing of the freshwater injections to the northern Atlantic that have been suggested as triggers for the global impacts associated with the Younger Dryas and Heinrich events. The second has to do with evidence for precursory events associated with the Heinrich ice-rafted debris layers in the northern Atlantic and with the abrupt Dansgaard-Oeschger warmings recorded in the Santa Barbara Basin.
NompC TRP Channel Required for Vertebrate Sensory Hair Cell Mechanotransduction
Authors: Sidi Samuel , Friedrich Rainer W. , Nicolson Teresa
Published: June 2003

The senses of hearing and balance in vertebrates rely on the sensory hair cells (HCs) of the inner ear. The central element of the HC's transduction apparatus is a mechanically gated ion channel of unknown identity. Here we report that the zebrafish ortholog of
Drosophila no mechanoreceptor potential C
(
nompC
), which encodes a transient receptor potential (TRP) channel, is critical for HC mechanotransduction. In zebrafish larvae,
nompC
is selectively expressed in sensory HCs. Morpholino-mediated removal of
nompC
function eliminated transduction-dependent endocytosis and electrical responses in HCs, resulting in larval deafness and imbalance. These observations indicate that
nompC
encodes a vertebrate HC mechanotransduction channel.
Laboratory Simulation of Charge Exchange-Produced X-ray Emission from Comets
Authors: Beiersdorfer P. , Boyce K. R. , Brown G. V. , Chen H. , Kahn S. M. , Kelley R. L. , May M. , Olson R. E. , Porter F. S. , Stahle C. K. , Tillotson W. A.
Published: June 2003

In laboratory experiments using the engineering spare microcalorimeter detector from the ASTRO-E satellite mission, we recorded the x-ray emission of highly charged ions of carbon, nitrogen, and oxygen, which simulates charge exchange reactions between heavy ions in the solar wind and neutral gases in cometary comae. The spectra are complex and do not readily match predictions. We developed a charge exchange emission model that successfully reproduces the soft x-ray spectrum of comet Linear C/1999 S4, observed with the Chandra X-ray Observatory.
Comprehensive Identification of Human bZIP Interactions with Coiled-Coil Arrays
Authors: Newman John R. S. , Keating Amy E.
Published: June 2003

In eukaryotes, the combinatorial association of sequence-specific DNA binding proteins is essential for transcription. We have used protein arrays to test 49
2
pairings of a nearly complete set of coiled-coil strands from human basic-region leucine zipper (bZIP) transcription factors. We find considerable partnering selectivity despite the bZIPs' homologous sequences. The interaction data are of high quality, as assessed by their reproducibility, reciprocity, and agreement with previous observations. Biophysical studies in solution support the relative binding strengths observed with the arrays. New associations provide insights into the circadian clock and the unfolded protein response.
Nod1 Detects a Unique Muropeptide from Gram-Negative Bacterial Peptidoglycan
Authors: Girardin Stephen E. , Boneca Ivo G. , Carneiro Leticia A. M. , Antignac Aude , Jéhanno Muguette , Viala Jérôme , Tedin Karsten , Taha Muhamed-Kheir , Labigne Agnès , Zäthringer Ulrich , Coyle Anthony J. , DiStefano Peter S. , Bertin John , Sansonetti Philippe J. , Philpott Dana J.
Published: June 2003

Although the role of Toll-like receptors in extracellular bacterial sensing has been investigated intensively, intracellular detection of bacteria through Nod molecules remains largely uncharacterized. Here, we show that human Nod1 specifically detects a unique diaminopimelate-containing
N
-acetylglucosamine–
N
-acetylmuramic acid (GlcNAc-MurNAc) tripeptide motif found in Gram-negative bacterial peptidoglycan, resulting in activation of the transcription factor NF-κB pathway. Moreover, we show that in epithelial cells (which represent the first line of defense against invasive pathogens), Nod1is indispensable for intracellular Gram-negative bacterial sensing.
Books Received
Authors: Landahl Andrew J.
Published: June 2003

A Shortcut Through Time
The Path to the Quantum Computer. George Johnson. Knopf, New York, 2003. 221 pp. $24, C$36. ISBN 0-375-41193-3.

In plain language, Johnson explains what quantum computers are and why we should care about them.
Estimating the Rock Volume Bias in Paleobiodiversity Studies
Authors: Crampton James S. , Beu Alan G. , Cooper Roger A. , Jones Craig M. , Marshall Bruce , Maxwell Phillip A.
Published: June 2003

To interpret changes in biodiversity through geological time, it is necessary first to correct for biases in sampling effort related to variations in the exposure of rocks and recovery of fossils with age. Data from New Zealand indicate that outcrop area is likely to be a reliable proxy of rock volume in both stable cratonic regions, where the paleobiodiversity record is strongly correlated with relative sea level, and on tectonically active margins. In contrast, another potential proxy, the number of rock formations, is a poor predictor of outcrop area or sampling effort in the New Zealand case.
Wnt Signaling, Ca
2
+
, and Cyclic GMP: Visualizing Frizzled Functions
Authors: Wang Hsien-yu , Malbon Craig C.
Published: June 2003

Wnts control the specification of cell fate, cell adhesion, migration, polarity, and proliferation. Their roles in development have been probed in fruit flies, nematodes, zebrafish, frogs, and mice. Some Wnts inhibit the degradation of β-catenin, which can regulate transcription of specific genes. Other Wnts exert their influences in other ways, such as increasing intracellular concentrations of Ca
2
+
and decreasing intracellular concentrations of cyclic guanosine monophosphate (cGMP). Heterotrimeric guanine nucleotide–binding proteins (G proteins) and RGS proteins have been implicated in Wnt signaling. Wnt regulation of intracellular Ca
2
+
and cGMP levels requires the G protein transducin and a cGMP-specific phosphodiesterase, which are major elements in signaling of the visual pathway.
The Signal to Move:
D. discoideum
Go Orienteering
Authors: Kimmel Alan R. , Parent Carole A.
Published: June 2003

Cells migrating directionally toward a chemoattractant source display a highly polarized cytoskeletal organization, with F-actin localized predominantly at the anterior and myosin II at the lateral and posterior regions.
Dictyostelium discoideum
has proven a useful system for elucidating signaling pathways that regulate this chemotactic response. During development, extracellular adenosine 3', 5' monophosphate (cAMP) functions as a primary signal to activate cell surface cAMP receptors (cARs). These receptors transduce different signals depending on whether or not they are coupled to heterotrimeric guanine nucleotide–binding proteins (G proteins) (see the STKE Connections Maps). Multiple G protein–stimulated pathways interact to establish polarity in chemotaxing
D. discoideum
cells by localizing F-actin at their leading edge and by regulating the phosphorylation state and assembly of myosin II. Many of the molecular interactions described are fundamental to the regulation of chemotaxis in other eukaryotic cells.
Mutants Suppressing Novobiocin Hypersensitivity of amukBNull Mutation
Authors: Adachi Shun , Hiraga Sota
Published: June 2003

ThemukBgene is essential for the partitioning of sister chromosomes inEscherichia coli. AmukBnull mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of themukBnull mutation. All suppressor mutations are localized in or near thegyrBgene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in themukBmutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.
Identification and Molecular Characterization of the Mg
2+
Stimulon of
Escherichia coli
Authors: Minagawa Shu , Ogasawara Hiroshi , Kato Akinori , Yamamoto Kaneyoshi , Eguchi Yoko , Oshima Taku , Mori Hirotada , Ishihama Akira , Utsumi Ryutaro
Published: June 2003

Transcription profile microarray analysis in
Escherichia coli
was performed to identify the member genes of the Mg
2+
stimulon that respond to the availability of external Mg
2+
in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl
2
, WP3022 (
phoP
defective), and WQ3007 (
phoQ
defective) were compared with those of W3110 in the absence of MgCl
2
. The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at
http://www.nara.kindai.ac.jp/nogei/seiken/array.html
), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg
2+
stimulon genes,
hemL
,
nagA
,
rstAB
,
slyB
,
vboR
, and
yrbL
, in addition to the
phoPQ
,
mgrB
, and
mgtA
genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the
phoPQ
,
mgrB
,
mgtA
,
hemL
,
nagA
,
rstAB
,
slyB
,
vboR
, and
yrbL
genes make up the Mg
2+
stimulon in
E
.
coli
.
mgr, a Novel Global Regulator inStaphylococcus aureus
Authors: Luong Thanh T. , Newell Steven W. , Lee Chia Y.
Published: June 2003

The virulence determinants ofStaphylococcus aureusare coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, namedmgr, was linked by transposon Tn917and mapped by three-factorial transduction crosses. The region containing themgrlocus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene,mgrA. Interestingly,mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream ofmgrAthat resulted in a four- to eightfold increase inmgrAtranscription in strain CYL5614. Thus, these results indicate thatmgrAis an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that themgrlocus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with theEscherichia coliMarR family of transcriptional regulators. Northern slot blot analyses suggested thatmgraffected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.
Electrophoretic Mobility of
Bacillus subtilis
Knockout Mutants with and without Flagella
Authors: Okuda Shujiro , Igarashi Ryosuke , Kusui Yusuke , Kasahara Yasuhiro , Morisaki Hisao
Published: June 2003

Mutants of
Bacillus subtilis
168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3. The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength. The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene. The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment. It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy. These results indicate that the flagella are a key factor influencing cell surface properties.
Repression of Phenazine Antibiotic Production in
Pseudomonas aureofaciens
Strain 30-84 by RpeA
Authors: Whistler Cheryl A. , Pierson Leland S.
Published: June 2003

Pseudomonas aureofaciens
strain 30-84 is a biological control bacterium that utilizes a two-component GacS/GacA regulatory system interconnected with the PhzR/PhzI quorum sensing system to positively regulate biosynthesis of phenazine antibiotics that contribute to its association with plant hosts. To date, no negative regulators of phenazine production have been identified, nor has the role of repression been studied. Here we describe a novel repressor of secondary metabolism in
P. aureofaciens
strain 30-84, RpeA, whose deduced amino acid sequence is similar to those of a group of putative two-component regulatory systems of unknown function found in several animal and plant-pathogenic bacteria. In minimal medium where phenazine production is very low, inactivation of the
rpeA
gene enhanced phenazine biosynthetic gene expression and increased phenazine production but did not increase quorum sensing signal accumulation. Furthermore, RpeA functioned to block phenazine biosynthetic gene transcription in minimal medium even when quorum-sensing signals were at a level that was sufficient for induction of phenazine gene expression in rich medium. Additionally, in the absence of
rpeA
, the quorum sensor PhzR was not required for phenazine production. Although repression plays a critical role in phenazine regulation, the
rpeA
mutation could not bypass the requirement for a functional GacS/GacA system, demonstrating that activation is required even in the absence of the RpeA repressor. This study reinforces that multiple signals, including nutrition and population density, are integrated to control the appropriate expression of phenazine antibiotics.
Probing the Catalytic Activity of a Cell Division-Specific Transpeptidase In Vivo with β-Lactams
Authors: Eberhardt Christian , Kuerschner Lars , Weiss David S.
Published: June 2003

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of
Escherichia coli
. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three β-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the
ftsA3
(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.
Molecular Analysis of Transport and Oligomerization of the
Yersinia enterocolitica
Adhesin YadA
Authors: Roggenkamp Andreas , Ackermann Nikolaus , Jacobi Christoph A. , Truelzsch Konrad , Hoffmann Harald , Heesemann Jürgen
Published: June 2003

The
Yersinia
adhesin YadA is the prototype of a novel class of bacterial adhesins which form oligomeric lollipop-like structures and are anchored in the outer membrane by the C terminus. For YadA, six different regions (R) or domains (D) are predicted from the amino acid sequence: the N-terminal leader sequence, head-D, neck-D, stalk-D, linking-R, and a C-terminal transmembrane region consisting of four β-strands. To identify structural and functional features of these domains, we performed in-frame deletion mutagenesis and constructed N-terminally tagged YadA variants. Diverse YadA variants were analyzed for outer membrane localization, surface exposure, oligomerization adhesion properties, and ability to protect against complement-mediated lysis. We demonstrated that (i) the C-terminal region (amino acids [aa] 353 to 422) is sufficient for outer membrane insertion and formation of trimers in the outer membrane; (ii) the head, neck, and stalk domains (aa 26 to 330) are surface exposed, forming a passenger domain; and (iii) the linking region (aa 331 to 369) is responsible for outer membrane translocation of the passenger domain. Thus, YadA meets all the criteria of an autotransporter. The same may be true for all other members of the YadA family, forming a subfamily of surface-attached oligomeric autotransporters. Moreover, in-frame truncation mutagenesis suggested that the head and neck domains together form the YadA-binding module which is located on the top of the stalk. However, the YadA-binding module did not confer serum resistance. Mutants lacking the head and neck domain were resistant to complement-mediated lysis. In-frame truncation of the stalk domain did not result in significant attenuation of the mutant in an orogastric mouse infection model.
Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in
Escherichia coli
K-12
Authors: Enz Sabine , Brand Heidi , Orellana Claudia , Mahren Susanne , Braun Volkmar
Published: June 2003

Transcription of the
fecABCDE
ferric citrate transport genes of
Escherichia coli
K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm. FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor. In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR
237-317
fragment interacts with the N-terminal FecA
1-79
fragment. In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis. They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins. The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium. Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E). One FecR mutant, FecR (D138E, V197A), induced
fecA
promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA. The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the
fec
transport genes and identified sites in FecR and FecA that are important for signal transduction.
Identification and Characterization of Transposable Elements of
Paracoccus pantotrophus
Authors: Bartosik Dariusz , Sochacka Marta , Baj Jadwiga
Published: June 2003

We studied diversity and distribution of transposable elements residing in different strains (DSM 11072, DSM 11073, DSM 65, and LMD 82.5) of a soil bacterium
Paracoccus pantotrophus
(α
-Proteobacteria
). With application of a shuttle entrapment vector pMEC1, several novel insertion sequences (ISs) and transposons (Tns) have been identified. They were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, terminal inverted repeats, as well as target sequences) and classification into the appropriate IS or Tn families. The frequency of transposition of these elements varied and ranged from 10
−6
to 10
−3
depending on the strain. The copy number, localization (plasmid or chromosome), and distribution of these elements in the
Paracoccus
species
P. pantotrophus
,
P. denitrificans
,
P. methylutens
,
P. solventivorans
, and
P. versutus
were analyzed. This allowed us to distinguish elements that are common in paracocci (IS
Ppa2
, IS
Ppa3
—both of the IS
5
family—and IS
Ppa5
of IS
66
family) as well as strain-specific ones (IS
Ppa1
of the IS
256
family, IS
Ppa4
of the IS
5
family, and Tn
3434
and Tn
5393
of the Tn
3
family), acquired by lateral transfer events. These elements will be of a great value in the design of new genetic tools for paracocci, since only one element (IS
1248
of
P. denitrificans
) has been described so far in this genus.
Cluster of Type IV Secretion Genes inHelicobacter pylori's Plasticity Zone
Authors: Kersulyte Dangeruta , Velapatiño Billie , Mukhopadhyay Asish K. , Cahuayme Lizbeth , Bussalleu Alejandro , Combe Juan , Gilman Robert H. , Berg Douglas E.
Published: June 2003

Some genes present in only certain strains of the genetically diverse gastric pathogenHelicobacter pylorimay affect its phenotype and/or evolutionary potential. Here we describe a new 16.3-kb segment, 7 of whose 16 open reading frames are homologs of type IV secretion genes (virB4,virB7tovirB11, andvirD4), the third such putative secretion gene cluster found inH. pylori. This segment, to be calledtfs3, was discovered by subtractive hybridization and chromosome walking. Full-length and truncatedtfs3elements were found in 20 and 19%, respectively, of 94 strains tested, which were from Spain, Peru, India, and Japan. Atfs3remnant (6 kb) was found in an archived stock of reference strain J99, although it was not included in this strain's published genome sequence. PCR and DNA sequence analyses indicated the following. (i)tfs3's ends are conserved. (ii) Right-end insertion occurred at one specific site in a chromosomal region that is varied in gene content and arrangement, the “plasticity zone.” (iii) Left-end insertion occurred at different sites in each of nine strains studied. (iv) Sequences next to the right-end target intfs3-free strains were absent from most strains carrying full-lengthtfs3elements. These patterns suggested insertion by a transposition-like event, but one in which targets are chosen with little or no specificity at the left end and high specificity at the right end, thereby deleting the intervening DNA.
Membrane Interaction of the Glycosyltransferase MurG: a Special Role for Cardiolipin
Authors: van den Brink-van der Laan Els , Boots Jan-Willem P. , Spelbrink Robin E. J. , Kool Gerda M. , Breukink Eefjan , Killian J. Antoinette , de Kruijff Ben
Published: June 2003

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of
Escherichia coli
MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from
E. coli
in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 ± 1 to 7 ± 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 ± 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.
Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
Authors: Ducote Matthew J. , Pettis Gregg S.
Published: June 2003

The conjugative plasmid pIJ101 of the spore-forming bacterium
Streptomyces lividans
contains a regulatory gene,
korB
, whose product is required to repress potentially lethal expression of the pIJ101
kilB
gene. The KorB protein also autoregulates
korB
gene expression and may be involved in control of pIJ101 copy number. KorB (pIJ101) is expressed as a 10-kDa protein in
S. lividans
that is immediately processed to a mature 6-kDa repressor molecule. The conjugative
Streptomyces cyanogenus
plasmid pSB24.1 is deleted upon entry into
S. lividans
to form pSB24.2, a nonconjugative derivative that contains a
korB
gene nearly identical to that of pIJ101. Previous evidence that
korB
of pSB24.2 is capable of overriding pIJ101
kilB
-associated lethality supported the notion that pIJ101 and pSB24.2 encode highly related, perhaps even identical conjugation systems. Here we show that KorB (pIJ101) and KorB (pSB24.2) repress transcription from the pIJ101
kilB
promoter equally well, although differences exist with respect to their interactions with
kilB
promoter sequences. Despite high sequence and functional similarities, KorB (pSB24.2) was found to exist as multiple stable forms ranging in size from 10 to 6 kDa both in
S. lividans
and
S. cyanogenus
. Immediate processing of KorB (pIJ101) exclusively to the 6-kDa repressor form meanwhile was conserved between the two species. A feature common to both proteins was a marked increase in expression or accumulation upon sporulation, an occurrence that may indicate a particular need for increased quantities of this regulatory protein upon spore germination and resumption of active growth of plasmid-containing cells.
Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes
Authors: York Gregory M. , Lupberger Joachim , Tian Jiamin , Lawrence Adam G. , Stubbe JoAnne , Sinskey Anthony J.
Published: June 2003

Intracellular poly[d-(−)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) fromRalstonia eutrophawas identified. We now report identification of candidate PHB depolymerase genes fromR.eutropha, namely,phaZ2andphaZ3, and their characterization in vivo.phaZ1was used to identify two candidate depolymerase genes in the genome ofRalstonia metallidurans.phaZ1and these genes were then used to design degenerate primers. These primers and PCR methods on theR.eutrophagenome were used to identify two new candidate depolymerase genes inR.eutropha:phaZ2andphaZ3. Inverse PCR methods were used to obtain the complete sequence ofphaZ3, and library screening was used to obtain the complete sequence ofphaZ2. PhaZ1, PhaZ2, and PhaZ3 share ∼30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generatingR.eutrophaH16 deletion strains (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ∼80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.
Identification and Mutational Analysis of Bacteriophage PRD1 Holin Protein P35
Authors: Rydman Pia S. , Bamford Dennis H.
Published: June 2003

Holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. We describe the identification of the membrane-containing phage PRD1 holin gene (gene
XXXV
). The PRD1 holin protein (P35, 12.8 kDa) acts similarly to its functional counterpart from phage lambda (gene
S
), and the defect in PRD1 gene
XXXV
can be corrected by the presence of gene
S
of lambda. Several nonsense, missense, and insertion mutations in PRD1 gene
XXXV
were analyzed. These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis.
Molecular Analysis of the Copper-Transporting Efflux System CusCFBA of
Escherichia coli
Authors: Franke Sylvia , Grass Gregor , Rensing Christopher , Nies Dietrich H.
Published: June 2003

The
cus
determinant of
Escherichia coli
encodes the CusCFBA proteins that mediate resistance to copper and silver by cation efflux. CusA and CusB were essential for copper resistance, and CusC and CusF were required for full resistance. Replacements of methionine residues 573, 623, and 672 with isoleucine in CusA resulted in loss of copper resistance, demonstrating their functional importance. Substitutions for several other methionine residues of this protein did not have any effect. The small 10-kDa protein CusF (previously YlcC) was shown to be a periplasmic protein. CusF bound one copper per polypeptide. The pink CusF copper protein complex exhibited an absorption maximum at around 510 nm. Methionine residues of CusF were involved in copper binding as shown by site-directed mutagenesis. CusF interacted with CusB and CusC polypeptides in a yeast two-hybrid assay. In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapartite resistance system that involves the novel periplasmic copper-binding protein CusF. These data provide additional evidence for the hypothesis that Cu(I) is directly transported from the periplasm across the outer membrane by the Cus complex.
Characterization of LytH, a Differentiation-Associated Peptidoglycan Hydrolase of
Bacillus subtilis
Involved in Endospore Cortex Maturation
Authors: Horsburgh Gavin J. , Atrih Abdelmadjid , Foster Simon J.
Published: June 2003

The cortex peptidoglycan from endospores of
Bacillus subtilis
is responsible for the maintenance of dormancy. LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination. The
lytH
gene was expressed only during sporulation, under the control of the mother cell-specific sigma factor σ
K
. Spores of a
lytH
mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy. Analysis of the peptidoglycan structure of
lytH
mutant spores shows the loss of muramic acid residues substituted with
l
-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain. In a
lytH cwlD
mutant, the lack of muramic acid residues substituted with
l
-alanine and δ-lactam leaves 97% of residues substituted with tetrapeptide. These results suggest that
lytH
encodes an
l
-Ala-
d
-Glu peptidase involved in production of single
l
-alanine side chains from tetrapeptides in the spore cortex. The lack of di- or tripeptides in a
lytH
mutant reveals the enzyme is an endopeptidase.
The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function
Authors: Enggist Elisabeth , Thöny-Meyer Linda
Published: June 2003

CcmE is a heme chaperone active in the cytochrome
c
maturation pathway of
Escherichia coli
. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome
c
. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type
c
cytochromes, only one additional residue, D131, was required, and for efficient cytochrome
c
maturation, the seven-residue sequence
131
DENYTPP
137
was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome
c
maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.
Characterization of the
Vibrio
vulnificus
putAP
Operon, Encoding Proline Dehydrogenase and Proline Permease, and Its Differential Expression in Response to Osmotic Stress
Authors: Lee Jeong Hyun , Park Na Young , Lee Myung Hee , Choi Sang Ho
Published: June 2003

The
Vibrio vulnificus putAP
genes encoding a proline dehydrogenase and a proline permease are transcribed in the same direction. Proline dehydrogenase activity and the level of
putA
transcript were determined to reach a maximum in exponential phase and were then repressed when growth slowed down. Northern blotting and primer extension analyses revealed that transcription of
putAP
genes results in two different transcripts, transcript A (
putA
transcript) and transcript AP (
putAP
transcript). Expression of
putAP
genes was directed by two promoters, promoter P

putA

and promoter P

putAP

. A
crp
null mutation decreased proline dehydrogenase activity and the level of the
put
transcripts, indicating that transcription of
putAP
is under the positive control of cyclic AMP receptor protein. Proline dehydrogenase and the level of both
put
transcripts were increased by proline but repressed by glutamate. In contrast, the level of transcript A, not transcript AP, increased when proline dehydrogenase was induced by NaCl. Since P

putA

activity, not P

putAP

activity, was increased by NaCl, it is apparent that transcript A and transcript AP are transcribed through P

putA

and P

putAP

, respectively. Cells challenged with NaCl and various hyperosmotic stresses accumulated higher levels of glutamate than control cells, indicating that glutamate is a compatible solute in
V
.
vulnificus
.
Striking Complexity of Lipopolysaccharide Defects in a Collection of
Sinorhizobium meliloti
Mutants
Authors: Campbell Gordon R. O. , Sharypova Larissa A. , Scheidle Heiko , Jones Kathryn M. , Niehaus Karsten , Becker Anke , Walker Graham C.
Published: June 2003

Although the role that lipopolysaccharide (LPS) plays in the symbiosis between
Sinorhizobium meliloti
and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood. This is largely due to a lack of mutants affected by its synthesis. In one of the definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on
Medicago sativa
, and concluded that LPS might not have a symbiotic function in
S. meliloti
. The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed. We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rm1021 and have characterized the resulting strains in greater detail. We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS. We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including
ddhB
,
lpsB
,
lpsC
, and
lpsE
. The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein. Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies. These results support more recent findings that alterations in LPS structure can affect the ability of
S. meliloti
to form an effective symbiosis.
Functional Characterization of Cysteine Residues in GlpT, the Glycerol 3-Phosphate Transporter of
Escherichia coli
Authors: Fann Mon-Chou , Busch Anne , Maloney Peter C.
Published: June 2003

In
Escherichia coli
, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration ≈ 35 μM) by the hydrophilic thiol-specific agent
p
-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes,
N
-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.
Suppressor Mutations in the Study of Photosystem I Biogenesis:
sll0088
Is a Previously Unidentified Gene Involved in Reaction Center Accumulation in
Synechocystis
sp. Strain PCC 6803
Authors: Yu Jianping , Shen Gaozhong , Wang Tao , Bryant Donald A. , Golbeck John H. , McIntosh Lee
Published: June 2003

In previous work, some members of our group isolated mutant strains of
Synechocystis
sp. strain PCC 6803 in which point mutations had been inserted into the
psaC
gene to alter the cysteine residues to the F
A
and F
B
iron-sulfur clusters in the PsaC subunit of photosystem I (J. P. Yu, I. R. Vassiliev, Y. S. Jung, J. H. Golbeck, and L. McIntosh, J. Biol. Chem.
272:
8032-8039, 1997). These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll
a
and photosystem I. In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 μmol m
−2
s
−1
). Two separate suppressor strains of C14S
PsaC
, termed C14S
PsaC
-R62 and C14S
PsaC
-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the
Synechocystis
sp. strain PCC 6803 genome named
sll0088
. C14S
PsaC
-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482→C change in
sll0088
, and C14S
PsaC
-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in
sll0088
. These suppressor strains showed near-wild-type levels of chlorophyll
a
and photosystem I, yet the serine oxygen ligand to F
B
was retained as shown by the retention of the
S
≥ 3/2 spin state of the [4Fe-4S] cluster. The inactivation of
sll0088
by insertion of a kanamycin resistance cartridge in the primary C14S
PsaC
mutant produced an engineered suppressor strain capable of photoautotrophic growth. There was no difference in
psaC
gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an
sll0088
deletion mutant. The
sll0088
gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms, including
Arabidopsis thaliana
. The protein contains a typical helix-turn-helix DNA-binding motif and can be classified as a negative regulator by phylogenetic analysis. This suggests that the product of
sll0088
has a role in regulating the biogenesis of photosystem I.
PAV1, the First Virus-Like Particle Isolated from a Hyperthermophilic Euryarchaeote, “
Pyrococcus abyssi
”
Authors: Geslin C. , Le Romancer M. , Erauso G. , Gaillard M. , Perrot G. , Prieur D.
Published: June 2003

We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of “
Pyrococcus abyssi
” previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm × 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the
Fuselloviridae
, isolated from
Sulfolobus shibatae
. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of
Thermococcales
isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.
Expression of the Nitroarene Dioxygenase Genes inComamonassp. Strain JS765 andAcidovoraxsp. Strain JS42 Is Induced by Multiple Aromatic Compounds
Authors: Lessner Daniel J. , Parales Rebecca E. , Narayan Shakti , Gibson David T.
Published: June 2003

This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) inComamonassp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) inAcidovoraxsp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes inPseudomonas putidaG7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from alacZreporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42ntdRnull mutant. Complementation with the closely related regulators NagR (fromRalstoniasp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes inPseudomonas putidaG7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.
Expression of
spoIIIJ
in the Prespore Is Sufficient for Activation of σ
G
and for Sporulation in
Bacillus subtilis
Authors: Serrano Mónica , Côrte Luísa , Opdyke Jason , Moran, Charles P. , Henriques Adriano O.
Published: June 2003

During sporulation in
Bacillus subtilis
, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor σ
F
. σ
F
directs transcription of
spoIIIG
, encoding the late forespore-specific regulator σ
G
. Following synthesis, σ
G
is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor. Activation of σ
G
occurs only after the complete engulfment of the prespore by the mother cell. Mutations in
spoIIIJ
arrest sporulation soon after conclusion of the engulfment process and prevent activation of σ
G
. Here we show that σ
G
accumulates but is mostly inactive in a
spoIIIJ
mutant. We also show that expression of the
spoIIIGE155K
allele, encoding a form of σ
G
that is not efficiently bound by SpoIIAB in vitro, restores σ
G
-directed gene expression to a
spoIIIJ
mutant. Expression of
spoIIIJ
occurs during vegetative growth. However, we show that expression of
spoIIIJ
in the prespore is sufficient for σ
G
activation and for sporulation. Mutations in the mother cell-specific
spoIIIA
locus are known to arrest sporulation just after completion of the engulfment process. Previous work has also shown that σ
G
accumulates in an inactive form in
spoIIIA
mutants and that the need for
spoIIIA
expression for σ
G
activation can be circumvented by the
spoIIIGE155K
allele. However, in contrast to the case for
spoIIIJ
, we show that expression of
spoIIIA
in the prespore does not support efficient sporulation. The results suggest that the activation of σ
G
at the end of the engulfment process involves the action of
spoIIIA
from the mother cell and of
spoIIIJ
from the prespore.