bluuebunny/crossref_metadata_2025 huggingface dataset:
PspG, a New Member of theYersinia enterocoliticaPhage Shock Protein Regulon
Authors: Green Rebecca C. , Darwin Andrew J.
Published: July 2004

TheYersinia enterocoliticaphage shock protein (Psp) system is induced when the Ysc type III secretion system is produced or when only the YscC secretin component is synthesized. Somepspnull mutants have a growth defect when YscC is produced and a severe virulence defect in animals. TheY. enterocolitica psplocus is made up of two divergently transcribed cistrons,pspFandpspABCDycjXF. pspAoperon expression is dependent on RpoN (σ54) and the enhancer-binding protein PspF. Previous data indicated that PspF also controls at least one gene that is not part of thepsplocus. In this study we describe the identification ofpspG, a new member of the PspF regulon. Predicted RpoN-binding sites upstream of thepspAgenes from different bacteria have a common divergence from the consensus sequence, which may be a signature of PspF-dependent promoters. TheY. enterocolitica pspGgene was identified because its promoter also has this signature. Like thepspAoperon,pspGis positively regulated by PspF, negatively regulated by PspA, and induced in response to the production of secretins. Purified His6-PspF protein specifically interacts with thepspAandpspGcontrol regions. ApspAoperon deletion mutant has a growth defect when the YscC secretin is produced and a virulence defect in a mouse model of infection. These phenotypes were exacerbated by apspGnull mutation. Therefore, PspG is the missing component of theY. enterocoliticaPsp regulon that was previously predicted to exist.
Systematic Mutagenesis of the
Escherichia coli
Genome
Authors: Kang Yisheng , Durfee Tim , Glasner Jeremy D. , Qiu Yu , Frisch David , Winterberg Kelly M. , Blattner Frederick R.
Published: July 2004

A high-throughput method has been developed for the systematic mutagenesis of the
Escherichia coli
genome. The system is based on in vitro transposition of a modified Tn
5
element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the λRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn
5
transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a
lacZ
allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn
5
complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn
5
transposition.
Fibronectin Binding to theSalmonella entericaSerotype Typhimurium ShdA Autotransporter Protein Is Inhibited by a Monoclonal Antibody Recognizing the A3 Repeat
Authors: Kingsley Robert A. , Abi Ghanem Daad , Puebla-Osorio Nahum , Keestra A. Marijke , Berghman Luc , Bäumler Andreas J.
Published: July 2004

ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of ∼1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin.
Two Biosynthetic Pathways for Aromatic Amino Acids in the Archaeon
Methanococcus maripaludis
Authors: Porat Iris , Waters Brian W. , Teng Quincy , Whitman William B.
Published: July 2004

Methanococcus maripaludis
is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 ± 0.2 mU mg
−1
in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and
p
-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-
13
C]phenylacetate.
M. maripaludis
has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate,
p
-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.
Characterization of the 3-
O
-Methylgallate Dioxygenase Gene and Evidence of Multiple 3-
O
-Methylgallate Catabolic Pathways in
Sphingomonas paucimobilis
SYK-6
Authors: Kasai Daisuke , Masai Eiji , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao
Published: July 2004

Sphingomonas paucimobilis
SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-
O
-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent
O
-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the
ligB
insertion mutant, which encodes the β subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the
desZ
gene, which confers 3MGA degradation activity on
Escherichia coli
. The deduced amino acid sequence of
desZ
showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both
desZ
and
ligB
in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the
desZ ligB
double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which
ligAB
,
desZ
, 3MGA
O
-demethylase, and gallate dioxygenase are participants.
Identification of the Protease and the Turnover Signal Responsible for Cell Cycle-Dependent Degradation of the
Caulobacter
FliF Motor Protein
Authors: Grünenfelder Björn , Tawfilis Sherif , Gehrig Stefanie , Østerås Magne , Eglin Daniel , Jenal Urs
Published: July 2004

Flagellar ejection is tightly coupled to the cell cycle in
Caulobacter crescentus
. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact
clpA
gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.
Circadian Rhythms in the Thermophilic Cyanobacterium
Thermosynechococcus elongatus
: Compensation of Period Length over a Wide Temperature Range
Authors: Onai Kiyoshi , Morishita Megumi , Itoh Shino , Okamoto Kazuhisa , Ishiura Masahiro
Published: July 2004

Proteins derived from the thermophilic cyanobacterium
Thermosynechococcus elongatus
BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium
T. elongatus
BP-1 that uses a bacterial luciferase gene set (Xl
luxAB
) derived from
Xenorhabdus luminescens
as a bioluminescence reporter gene. A promoter region of the
psbA1
gene of
T. elongatus
was fused to the Xl
luxAB
gene set and inserted into a specific targeting site in the genome of
T. elongatus
. The bioluminescence from the cells of the
psbA1
-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60°C). Theses results indicate that
T. elongatus
has the circadian clock that is widely temperature compensated.
6S RNA Function Enhances Long-Term Cell Survival
Authors: Trotochaud Amy E. , Wassarman Karen M.
Published: July 2004

6S RNA was identified in
Escherichia coli
>30 years ago, but the physiological role of this RNA has remained elusive. Here, we demonstrate that 6S RNA-deficient cells are at a disadvantage for survival in stationary phase, a time when 6S RNA regulates transcription. Growth defects were most apparent as a decrease in the competitive fitness of cells lacking 6S RNA. To decipher the molecular mechanisms underlying the growth defects, we have expanded studies of 6S RNA effects on transcription. 6S RNA inhibition of σ
70
-dependent transcription was not ubiquitous, in spite of the fact that the vast majority of σ
70
-RNA polymerase is bound by 6S RNA during stationary phase. The σ
70
-dependent promoters inhibited by 6S RNA contain an extended −10 promoter element, suggesting that this feature may define a class of 6S RNA-regulated genes. We also discovered a secondary effect of 6S RNA in the activation of σ
S
-dependent transcription at several promoters. We conclude that 6S RNA regulation of both σ
70
and σ
S
activities contributes to increased cell persistence during nutrient deprivation.
The Global Regulator Genes from Biocontrol Strain
Serratia plymuthica
IC1270: Cloning, Sequencing, and Functional Studies
Authors: Ovadis Marianna , Liu Xiaoguang , Gavriel Sagi , Ismailov Zafar , Chet Ilan , Chernin Leonid
Published: July 2004

The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than
Pseudomonas
spp.,
gacA/gacS
and
rpoS
homologues were cloned from biocontrol strain IC1270 of
Serratia plymuthica
, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned
gacA/gacS-
like genes
—
tentatively designated
grrA
(global response regulation activator) and
grrS
(global response regulation sensor) and of the cloned
rpoS
gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria
Escherichia coli
,
Pectobacterium carotovora
subsp.
carotovora
, and
Serratia marcescens. grrA
,
grrS
, and
rpoS
gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and
N
-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only
grrA
and
grrS
mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the
grrA
,
grrS
, and
rpoS
mutants were markedly less capable of suppressing
Rhizoctonia solani
and
Pythium aphanidermatum
under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.
Intraspecific Phylogeny and Lineage Group Identification Based on theprfAVirulence Gene Cluster ofListeria monocytogenes†
Authors: Ward Todd J. , Gorski Lisa , Borucki Monica K. , Mandrell Robert E. , Hutchins Jan , Pupedis Kitty
Published: July 2004

Listeria monocytogenesis a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regardingL.monocytogeneslineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based onprfAvirulence gene cluster sequences from 113L.monocytogenesisolates. The results of the multigene phylogenetic analyses confirm thatL. monocytogenescomprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the threeL. monocytogeneslineages may represent distinct species within the framework of the cohesion species concept.
Replication of
Enterococcus faecalis
Pheromone-Responding Plasmid pAD1: Location of the Minimal Replicon and
oriV
Site and RepA Involvement in Initiation of Replication
Authors: Francia Maria Victoria , Fujimoto Shuhei , Tille Patricia , Weaver Keith E. , Clewell Don B.
Published: July 2004

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of
Enterococcus faecalis
. The determinants
repA
,
repB
, and
repC
, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The
repA
gene encodes an initiator protein, whereas
repB
and
repC
encode proteins related to stability and copy number. The present study focuses specifically on
repA
and identifies a replication origin (
oriV
) within a central region of the
repA
determinant. A small segment of
repA
carrying
oriV
was able to support replication in
cis
of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in
trans
. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only
repA
is sufficient for stable replication in
E. faecalis
. We also show that RepA binds specifically to
oriV
DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize
oriV
. An in-frame deletion within
repA
resulting in loss of 105 nucleotides, including at least part of
oriV
, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in
trans
. The relationship of RepA to other known initiator proteins is also discussed.
Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in
Mycobacterium tuberculosis
as It Goes into a Dormancy-Like State in Culture
Authors: Daniel Jaiyanth , Deb Chirajyoti , Dubey Vinod S. , Sirakova Tatiana D. , Abomoelak Bassam , Morbidoni Hector R. , Kolattukudy Pappachan E.
Published: July 2004

Mycobacterium tuberculosis
enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in
M. tuberculosis
. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (
tgs
) when expressed in
Escherichia coli
showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several
tgs
genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O
2
and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in
E. coli
shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.
A Type II Protein Secretory Pathway Required for Levansucrase Secretion by
Gluconacetobacter diazotrophicus
Authors: Arrieta Juan G. , Sotolongo Mailin , Menéndez Carmen , Alfonso Dubiel , Trujillo Luis E. , Soto Melvis , Ramírez Ricardo , Hernández Lázaro
Published: July 2004

The endophytic diazotroph
Gluconacetobacter diazotrophicus
secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated
lsdX
, is located between the operon promoter and
lsdG
, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The
lsdX
,
-G
, and -
O
genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes
lsdE
,
-F
,
-H
,
-I
,
-J
, -
L
,
-M
,
-N
, and -
D
were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard
G. diazotrophicus
structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of
lsdG
,
lsdO
, or
lsdF
blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys
162
by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (
lsdA
-
lsdB
) locus in 14
G. diazotrophicus
strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the
Acetobacteraceae
.
Comparative Whole-Genome Hybridization Reveals Genomic Islands inBrucellaSpecies
Authors: Rajashekara Gireesh , Glasner Jeremy D. , Glover David A. , Splitter Gary A.
Published: July 2004

Brucellaspecies are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species.Brucella abortus,B. melitensis, andB. suisare pathogenic to humans, whereasB. ovisandB. neotomaeare nonpathogenic to humans andB. canishuman infections are rare. Limited genome diversity exists amongBrucellaspecies. Comparison ofBrucellaspecies whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence ofB. melitensis16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA fromBrucellaspecies to this microarray revealed a total of 217 open reading frames (ORFs) altered in fiveBrucellaspecies analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified inBrucellaspecies are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certainBrucellaspecies for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.
Three-Dimensional Electron Microscopic Imaging of Membrane Invaginations in
Escherichia coli
Overproducing the Chemotaxis Receptor Tsr
Authors: Lefman Jonathan , Zhang Peijun , Hirai Teruhisa , Weis Robert M. , Juliani Jemma , Bliss Donald , Kessel Martin , Bos Erik , Peters Peter J. , Subramaniam Sriram
Published: July 2004

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of
Escherichia coli
cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type
E. coli
and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.
The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of
l
-Phenylalanine,
l
-Tyrosine, and 3-Hydroxyphenylacetate in
Pseudomonas putida
Authors: Arias-Barrau Elsa , Olivera Elías R. , Luengo José M. , Fernández Cristina , Galán Beatriz , García José L. , Díaz Eduardo , Miñambres Baltasar
Published: July 2004

Pseudomonas putida
metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the
phh
,
tyr
, and
hpd
genes are not linked in the
P. putida
genome, the
hmgABC
genes appear to form a single transcriptional unit. Gel retardation assays and
lacZ
translational fusion experiments have shown that
hmgR
encodes a specific repressor that controls the inducible expression of the divergently transcribed
hmgABC
catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the
Phmg
promoter that spans a 17-bp palindromic motif and an external direct repetition from position −16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the
hmgABC
,
hpd
, and
tyrB
genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in
P. putida
U funnels also into the homogentisate central pathway, revealing that the
hmg
cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.
Secretion and Function ofSalmonellaSPI-2 Effector SseF Require Its Chaperone, SscB
Authors: Dai Shipan , Zhou Daoguo
Published: July 2004

Salmonellastrains utilize a type III secretion system for their successful survival and replications inside host cells. SseF is one of the several effector proteins that are required for conferring this survival ability by altering the trafficking of theSalmonella-containing vacuoles. These effector proteins often require appropriate chaperones to maintain their stabilities inside the bacteria. These chaperones are also known to assist the subsequent secretion and translocation of their substrates. We report here that SscB acts as the chaperone for SseF, an effector for theSalmonellapathogenicity island 2 (SPI-2). We found that thesscBgene is required for the formation ofSalmonellasp.-induced continuous filaments in epithelial cells. EfficientSalmonellareplication in macrophages requires SscB function. Intracellular and secretion levels of SseF are greatly reduced in ansscBmutant strain compared to the wild-type strain. A protein stability assay demonstrated that the half-life of SseF is significantly shortened in the absence of SscB. Transcriptional analysis of thesseFgene showed that the effect of SscB on the SseF level is not at the transcriptional level. A coprecipitation experiment indicated that SscB interacts with SseF. In summary, our results indicate that SscB is a chaperone for SPI-2 effector SseF to facilitate its secretion and function inside the host cells.
A Movable Surface: Formation of
Yersinia
sp. Biofilms on Motile
Caenorhabditis elegans
Authors: Tan Li , Darby Creg
Published: July 2004

Bubonic plague is transmitted by fleas whose feeding is blocked by a mass of
Yersinia pestis
in the digestive tract.
Y. pestis
and the closely related
Y. pseudotuberculosis
also block the feeding of
Caenorhabditis elegans
by forming a biofilm on the nematode head.
C. elegans
mutants with severe motility defects acquire almost no biofilm, indicating that normal animals accumulate the biofilm matrix as they move through a
Yersinia
lawn. Using the lectin wheat germ agglutinin as a probe, we show that the matrix on
C. elegans
contains carbohydrate produced by
Yersinia
. The carbohydrate is present in bacterial lawns prior to addition of nematodes, indicating that biofilm formation does not involve signaling between the two organisms. Furthermore, biofilm accumulation depends on continuous
C. elegans
exposure to a lawn of
Yersinia
bacteria.
A Second Lysine-Specific Serine Protease from
Lysobacter
sp. Strain IB-9374
Authors: Chohnan Shigeru , Shiraki Kentaro , Yokota Kiyonobu , Ohshima Makoto , Kuroiwa Natsuki , Ahmed Kashfia , Masaki Takeharu , Sakiyama Fumio
Published: July 2004

A second lysyl endopeptidase gene (
lepB
) was found immediately upstream of the previously isolated
lepA
gene encoding a highly active lysyl endopeptidase in
Lysobacter
genomic DNA. The
lepB
gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the
lepA
and
lepB
gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The
lepB
gene encoding the propeptide and mature-enzyme portions was overexpressed in
Escherichia coli
, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.
Nucleotide Sequence and Evolution of the Five-Plasmid Complement of the Phytopathogen
Pseudomonas syringae
pv. maculicola ES4326
Authors: Stavrinides John , Guttman David S.
Published: July 2004

Plasmids are transmissible, extrachromosomal genetic elements that are often responsible for environmental or host-specific adaptations. In order to identify the forces driving the evolution of these important molecules, we determined the complete nucleotide sequence of the five-plasmid complement of the radish and
Arabidopsis
pathogen
Pseudomonas syringae
pv. maculicola ES4326 and conducted an intraspecific comparative genomic analysis. To date, this is the most complex fully sequenced plasmid complement of any gram-negative bacterium. The plasmid complement comprises two pPT23A-like replicons, pPMA4326A (46,697 bp) and pPMA4326B (40,110 bp); a pPS10-like replicon, pPMA4326C (8,244 bp); and two atypical, replicase-deficient replicons, pPMA4326D (4,833 bp) and pPMA4326E (4,217 bp). A complete type IV secretion system is found on pPMA4326A, while the type III secreted effector
hopPmaA
is present on pPMA4326B. The region around
hopPmaA
includes a shorter
hopPmaA
homolog, insertion sequence (IS) elements, and a three-element cassette composed of a resolvase, an integrase, and an
exeA
gene that is also present in several human pathogens. We have also identified a novel genetic element (E622) that is present on all but the smallest plasmid (pPMA4326E) that has features of an IS element but lacks an identifiable transposase. This element is associated with virulence-related genes found in a wide range of
P. syringae
strains. Comparative genomic analyses of these and other
P. syringae
plasmids suggest a role for recombination and integrative elements in driving plasmid evolution.
CloningSerratia entomophilaAntifeeding Genes—a Putative Defective Prophage Active against the Grass GrubCostelytra zealandica
Authors: Hurst Mark R. H. , Glare Travis R. , Jackson Trevor A.
Published: July 2004

Serratia entomophilaandSerratia proteamaculans(Enterobacteriaceae) cause amber disease in the grass grubCostelytra zealandica(Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 155-kb plasmid, pADAP, carries the genessepA,sepB, andsepC, which are essential for production of amber disease symptoms. Transposon insertions in any of thesepgenes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either anEscherichia colior aSerratiabackground exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present inPhotorhabdus luminescenssubsp.laumondiiTTO1, a species which also hassepgene homologues.
The Phosphoenolpyruvate Carboxylase from
Methanothermobacter thermautotrophicus
Has a Novel Structure
Authors: Patel Hiten M. , Kraszewski Jessica L. , Mukhopadhyay Biswarup
Published: July 2004

In
Methanothermobacter thermautotrophicus
, oxaloacetate synthesis is a major and essential CO
2
-fixation reaction. This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase. The phosphoenolpyruvate carboxylase from this organism was purified to homogeneity. The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (PPCs). The NH
2
-terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence. A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved. This is the first report of a new type of phosphoenolpyruvate carboxylase that we call PpcA (“A” for “archaeal”). Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (
Clostridium perfringens
,
Oenococcus oeni
, and
Leuconostoc mesenteroides
) and no eukaryotic genomes. PpcA was the only recognizable oxaloacetate-producing enzyme in
Methanopyrus kandleri
, a hydrothermal vent organism. Each PpcA-containing organism lacked a PPC homolog. The activity of
M. thermautotrophicus
PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the
Escherichia coli
PPC. The catalytic core (including His
138
, Arg
587
, and Gly
883
) of the
E. coli
PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys
773
, Arg
832
, and Asn
881
) were not. PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme. The reverse is also equally possible.
DNA Microarray Analysis of Genome Dynamics in
Yersinia pestis
: Insights into Bacterial Genome Microevolution and Niche Adaptation
Authors: Zhou Dongsheng , Han Yanping , Song Yajun , Tong Zongzhong , Wang Jin , Guo Zhaobiao , Pei Decui , Pang Xin , Zhai Junhui , Li Min , Cui Baizhong , Qi Zhizhen , Jin Lixia , Dai Ruixia , Du Zongmin , Bao Jingyue , Zhang Xiuqing , Yu Jun , Wang Jian , Huang Peitang , Yang Ruifu
Published: July 2004

Genomics research provides an unprecedented opportunity for us to probe into the pathogenicity and evolution of the world's most deadly pathogenic bacterium,
Yersinia pestis
, in minute detail. In our present work, extensive microarray analysis in conjunction with PCR validation revealed that there are considerable genome dynamics, due to gene acquisition and loss, in natural populations of
Y. pestis
. We established a genomotyping system to group homologous isolates of
Y. pestis
, based on profiling or gene acquisition and loss in their genomes, and then drew an outline of parallel microevolution of the
Y. pestis
genome. The acquisition of a number of genomic islands and plasmids most likely induced
Y. pestis
to evolve rapidly from
Yersinia pseudotuberculosis
to a new, deadly pathogen. Horizontal gene acquisition also plays a key role in the dramatic evolutionary segregation of
Y. pestis
lineages (biovars and genomovars). In contrast to selective genome expansion by gene acquisition, genome reduction occurs in
Y. pestis
through the loss of DNA regions. We also theorized about the links between niche adaptation and genome microevolution. The transmission, colonization, and expansion of
Y. pestis
in the natural foci of endemic plague are parallel and directional and involve gradual adaptation to the complex of interactions between the environment, the hosts, and the pathogen itself. These adaptations are based on the natural selections against the accumulation of genetic changes within genome. Our data strongly support that the modern plague originated from Yunnan Province in China, due to the arising of biovar orientalis from biovar antiqua rather than mediaevalis.
Genetics of Metabolic Variations between
Yersinia pestis
Biovars and the Proposal of a New Biovar, microtus
Authors: Zhou Dongsheng , Tong Zongzhong , Song Yajun , Han Yanping , Pei Decui , Pang Xin , Zhai Junhui , Li Min , Cui Baizhong , Qi Zhizhen , Jin Lixia , Dai Ruixia , Du Zongmin , Wang Jin , Guo Zhaobiao , Wang Jian , Huang Peitang , Yang Ruifu
Published: July 2004

Yersinia pestis
has been historically divided into three biovars: antiqua, mediaevalis, and orientalis. On the basis of this study, strains from
Microtus
-related plague foci are proposed to constitute a new biovar, microtus. Based on the ability to ferment glycerol and arabinose and to reduce nitrate,
Y. pestis
strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative). A 93-bp in-frame deletion in
glpD
gene results in the glycerol-negative characteristic of biovar orientalis strains. Two kinds of point mutations in the
napA
gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively. A 122-bp frameshift deletion in the
araC
gene may lead to the arabinose-negative phenotype of biovar microtus strains. Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar. Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of
Y. pestis
and plague pathogenesis. Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine.
PBP1 Is a Component of theBacillus subtilisCell Division Machinery
Authors: Scheffers Dirk-Jan , Errington Jeffery
Published: July 2004

Bacillus subtilispenicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of theB. subtiliscell division machinery.
Cellular Levels of
trp
RNA-Binding Attenuation Protein in
Bacillus subtilis
Authors: McCabe Barbara C. , Gollnick Paul
Published: July 2004

Expression of the
Bacillus subtilis trp
genes is negatively regulated by an 11-subunit
trp
RNA-binding attenuation protein (TRAP), which is activated to bind RNA by binding
l
-tryptophan. We used Western blotting to estimate that there are 200 to 400 TRAP 11-mer molecules per cell in cells grown in either minimal or rich medium.
Expression of a Functional Secreted YopN-TyeA Hybrid Protein inYersinia pestisIs the Result of a +1 Translational Frameshift Event
Authors: Ferracci Franco , Day James B. , Ezelle Heather J. , Plano Gregory V.
Published: July 2004

YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA. This study identifies a secreted YopN-TyeA hybrid protein that is expressed byYersinia pestis, but not byYersinia enterocolitica. DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event.
The
ompU
Paralogue
vca1008
Is Required for Virulence of
Vibrio cholerae
Authors: Osorio Carlos G. , Martinez-Wilson Hector , Camilli Andrew
Published: July 2004

We made single and combined mutations in
ompU
,
ompT
, and the two putative porin genes
vca1008
and
vc0972
. The fitness of the strains was tested in vitro and in the infant mouse model of intestinal infection. We also studied the transcriptional induction of
vca1008
in vitro and during mouse infection. We show that
vca1008
is induced during infection and is necessary and sufficient (in the absence of
ompU
,
ompT
, and
vc0972
) for infection.
Biochemical Study of Multiple CheY Response Regulators of the Chemotactic Pathway of
Rhodobacter sphaeroides
Authors: Ferré Axelle , de la Mora Javier , Ballado Teresa , Camarena Laura , Dreyfus Georges
Published: July 2004

The six copies of the response regulator CheY from
Rhodobacter sphaeroides
bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac
32
P was observed in five CheY proteins. We also show that all of the
cheY
genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.
Polyphosphate Synthetic Activity of Polyphosphate:AMP Phosphotransferase in
Acinetobacter johnsonii
210A
Authors: Itoh Hiromichi , Shiba Toshikazu
Published: July 2004

Polyphosphate:AMP phosphotransferase (PAP) has been identified as an enzyme that catalyzes the phosphorylation of AMP with inorganic polyphosphates [poly(P)] as phosphate donors. We found that the purified PAP of
Acinetobacter johnsonii
210A has poly(P) synthetic activity. The PAP catalyzes the dephosphorylation of ADP and processively synthesizes poly(P) of 200 to 700 residues. Comparatively lower concentrations of MgCl
2
(20 mM) were required to obtain optimum poly(P) synthetic activity, whereas higher concentrations of MgCl
2
(100 mM) were necessary for optimum PAP activity. ADP is preferred over GDP as a phosphate donor for poly(P) synthesis. The

K
m

and
V
max
values for ADP in the poly(P) synthetic activity of PAP were 8.3 mM and 55 μmol min
−1
mg
−1
, respectively. We concluded that the PAP of
A. johnsonii
210A is a novel type of poly(P) kinase that uses ADP and GDP as substrates.
Evolution of the
Helicobacter pylori
Vacuolating Cytotoxin in a Human Stomach
Authors: Aviles-Jimenez Francisco , Letley Darren P. , Gonzalez-Valencia Gerardo , Salama Nina , Torres Javier , Atherton John C.
Published: July 2004

We describe two subclones of
Helicobacter pylori
, isolated contemporaneously from a human stomach, which differ markedly in the vacuolating cytotoxin gene,
vacA
, but whose near identity in sequences outside this locus implies a very recent common origin. The differences are consistent with homologous recombination with DNA from another strain and result in a changed
vacA
midregion and, importantly, in changed toxicity.
GENERALIZED MAHLER MEASURES AND MULTIPLE SINE FUNCTIONS
Authors: GON YASURO , OYANAGI HIDEO
Published: July 2004

We introduce a generalized Mahler measure. It has relations to multiple sine functions and Dirichlet L-functions. In particular, we are able to express special values of Dirichlet L-functions by sum of logarithmic generalized Mahler measures.
RESIDUES FOR SINGULAR PAIRS AND DYNAMICS OF BIHOLOMORPHIC MAPS OF SINGULAR SURFACES
Authors: BRACCI FILIPPO , SUWA TATSUO
Published: July 2004

We prove the existence of a parabolic curve for a germ of biholomorphic map tangent to the identity at an isolated singular point of a surface under some conditions. For this purpose, we present a Camacho–Sad type index theorem for fixed curves of biholomorphic maps of singular surfaces and develop a local intersection theory of curves in singular surfaces from an analytic approach by means of Grothendieck residues.
APPROXIMATIONS OF HOLOMORPHIC FUNCTIONS IN CERTAIN BANACH SPACES
Authors: JOSEFSON BENGT
Published: July 2004

Let E be a Banach space and let B(R)⊂E denote the open ball with centre at 0 and radius R. The following problem is studied: given 0<r0 and a function f holomorphic on B(R), does there always exist an entire function g on E such that |f-g|<∊ on B(r)?

L. Lempert has proved that the answer is positive for Banach spaces having an unconditional basis with unconditional constant 1. In this paper a somewhat shorter proof of Lemperts result is given.

In general it is not possible to approximate f by polynomials since f does not need to be bounded on B(r).