bluuebunny/crossref_metadata_2025 huggingface dataset:
Trichomoniasis
Authors: Schwebke Jane R. , Burgess Donald
Published: October 2004

Trichomoniasis is perhaps the most common curable sexually transmitted disease worldwide, yet few resources are devoted to its control. It is associated with potentially serious complications such as preterm birth and human immunodeficiency virus acquisition and transmission. The immunology of a related organism, Tritrichomonas foetus, which causes disease in cattle, has been investigated to some extent, but more work is needed for the human strain, Trichomonas vaginalis. In addition, although trichomoniasis is easily treated with oral metronidazole, there is concern that the number of strains resistant to this antibiotic are increasing, and currently no alternative is licensed in the United States. As more is appreciated concerning the important public health implications of this common infection, more work will need to be done in understanding the diagnosis, treatment, and immunology of this organism.
Sarcocystis
spp. in Human Infections
Authors: Fayer Ronald
Published: October 2004

Sarcocystis
species are intracellular protozoan parasites with an intermediate-definitive host life cycle based on a prey-predator relationship. Asexual stages develop in intermediate hosts after they ingest the oocyst stage from definitive-host feces and terminate with the formation of intramuscular cysts (sarcocysts). Sarcocysts in meat eaten by a definitive host initiate sexual stages in the intestine that terminate in oocysts excreted in the feces. Most
Sarcocystis
species infect specific hosts or closely related host species. For example, humans and some primates are definitive hosts for
Sarcocystis hominis
and
S. suihominis
after eating raw meat from cattle and pigs, respectively. The prevalence of intestinal sarcocystosis in humans is low and is only rarely associated with illness, except in volunteers who ingest large numbers of sarcocysts. Cases of infection of humans as intermediate hosts, with intramuscular cysts, number less than 100 and are of unknown origin. The asexual stages, including sarcocysts, can stimulate a strong inflammatory response. Livestock have suffered acute debilitating infections, resulting in abortion and death or chronic infections with failure to grow or thrive. This review provides a summary of
Sarcocystis
biology, including its morphology, life cycle, host specificity, prevalence, diagnosis, treatment, and prevention strategies, for human and food animal infections.
The Molecular Mechanisms Used byNeisseria gonorrhoeaeTo Initiate Infection Differ between Men and Women
Authors: Edwards Jennifer L. , Apicella Michael A.
Published: October 2004

The molecular mechanisms used by the gonococcus to initiate infection exhibit gender specificity. The clinical presentations of disease are also strikingly different upon comparison of gonococcal urethritis to gonococcal cervicitis. An intimate association occurs between the gonococcus and the urethral epithelium and is mediated by the asialoglycoprotein receptor. Gonococcal interaction with the urethral epithelia cell triggers cytokine release, which promotes neutrophil influx and an inflammatory response. Similarly, gonococcal infection of the upper female genital tract also results in inflammation. Gonococci invade the nonciliated epithelia, and the ciliated cells are subjected to the cytotoxic effects of tumor necrosis factor alpha induced by gonococcal peptidoglycan and lipooligosaccharide. In contrast, gonococcal infection of the lower female genital tract is typically asymptomatic. This is in part the result of the ability of the gonococcus to subvert the alternative pathway of complement present in the lower female genital tract. Gonococcal engagement of complement receptor 3 on the cervical epithelia results in membrane ruffling and does not promote inflammation. A model of gonococcal pathogenesis is presented in the context of the male and female human urogenital tracts.
Diagnosis and Assessment of Trachoma
Authors: Solomon Anthony W. , Peeling Rosanna W. , Foster Allen , Mabey David C. W.
Published: October 2004

Trachoma is caused byChlamydia trachomatis. Clinical grading with the WHO simplified system can be highly repeatable provided graders are adequately trained and standardized. At the community level, rapid assessments are useful for confirming the absence of trachoma but do not determine the magnitude of the problem in communities where trachoma is present. New rapid assessment protocols incorporating techniques for obtaining representative population samples (without census preparation) may give better estimates of the prevalence of clinical trachoma. Clinical findings do not necessarily indicate the presence or absence ofC. trachomatisinfection, particularly as disease prevalence falls. The prevalence of ocularC. trachomatisinfection (at the community level) is important because it is infection that is targeted when antibiotics are distributed in trachoma control campaigns. Methods to estimate infection prevalence are required. While culture is a sensitive test for the presence of viable organisms and nucleic acid amplification tests are sensitive and specific tools for the presence of chlamydial nucleic acids, the commercial assays presently available are all too expensive, too complex, or too unreliable for use in national programs. There is an urgent need for a rapid, reliable test forC. trachomatisto assist in measuring progress towards the elimination of trachoma.
Genome of Bacteriophage P1
Authors: Łobocka Małgorzata B. , Rose Debra J. , Plunkett Guy , Rusin Marek , Samojedny Arkadiusz , Lehnherr Hansjörg , Yarmolinsky Michael B. , Blattner Frederick R.
Published: October 2004

P1 is a bacteriophage ofEscherichia coliand other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from σ70promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by theE. coliRNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.
Lactobacillus plantarum
Bacteriophage LP65: a New Member of the SPO1-Like Genus of the Family
Myoviridae
Authors: Chibani-Chennoufi Sandra , Dillmann Marie-Lise , Marvin-Guy Laure , Rami-Shojaei Sabrina , Brüssow Harald
Published: October 2004

The virulent
Lactobacillus plantarum
myophage LP65 was isolated from industrial meat fermentation. Tail contraction led to reorganization of the tail sheath and the baseplate; a tail tube was extruded. In ultrathin section the phage adsorbed via its baseplate to the exterior of the cell, while the tail tube tunneled through the thick bacterial cell wall. Convoluted membrane structures were induced in the infected cell. Progeny phage was detected 100 min postinfection, and lysis occurred after extensive digestion of the cell wall. Sequence analysis revealed a genome of 131,573 bp of nonredundant DNA. Four major genome regions and a large tRNA gene cluster were observed. One module corresponded to DNA replication genes. Helicase/primase and two replication/recombination enzymes represented the only links to T4-like
Myoviridae
from gram-negative bacteria. Another module corresponded to the structural genes. Sequence relatedness identified links with
Listeria
phage A511,
Staphylococcus
phage K, and
Bacillus
phage SPO1. LP65 structural proteins were identified by two-dimensional proteome analysis and mass spectrometry. The putative tail sheath protein showed a shear-induced change in electrophoretic migration behavior. The genome organization of the structural module in LP65 resembled that of
Siphoviridae
from the lambda supergroup.
Multicopy Plasmids Affect Replisome Positioning inBacillus subtilis
Authors: Wang Jue D. , Rokop Megan E. , Barker Melanie M. , Hanson Nathaniel R. , Grossman Alan D.
Published: October 2004

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells ofBacillus subtilisusing an array oflacoperators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that inB. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location oforiCat the time of initiation of replication.
Rgg Regulates Growth Phase-Dependent Expression of Proteins Associated with Secondary Metabolism and Stress inStreptococcus pyogenes
Authors: Chaussee Michelle A. , Callegari Eduardo A. , Chaussee Michael S.
Published: October 2004

The transcriptional regulatory protein Rgg coordinates amino acid catabolism and virulence factor expression inStreptococcus pyogenes. We used a proteomic approach to compare cytoplasmic proteins isolated fromS. pyogeneswild-type strain NZ131 (serotype M49) to proteins isolated from anrggmutant strain during the exponential and stationary phases of growth. Proteins were separated by two-dimensional gel electrophoresis, and 125 protein spots of interest were identified by tandem mass spectrometry. Comparative analysis of proteins isolated from the isogenic strains revealed that growth phase-associated regulation of enzymes involved in the metabolism of arginine (ArcABC), histidine (HutI), and serine (SdhA) was abrogated in therggmutant strain, which synthesized the proteins in the exponential phase of growth. In contrast, the enzymes were detected only among wild-type proteins isolated from organisms in the stationary phase of growth. The differences in protein composition were correlated with previously described metabolic changes. In addition, proteins associated with thermal and oxidative stress responses, including ClpE and ClpL, were present in samples isolated from therggmutant strain but not in samples isolated from the wild-type strain. Therggmutant strain was more tolerant to elevated temperature and puromycin than the wild-type strain; however, the mutant was less tolerant to paraquat. We concluded that Rgg is a global regulatory factor that contributes to growth phase-dependent synthesis of proteins associated with secondary metabolism and oxidative and thermal stress responses.
Four Proteins Encoded in the
gspB-secY2A2
Operon of
Streptococcus gordonii
Mediate the Intracellular Glycosylation of the Platelet-Binding Protein GspB
Authors: Takamatsu Daisuke , Bensing Barbara A. , Sullam Paul M.
Published: October 2004

Platelet binding by
Streptococcus gordonii
strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the
secY2A2
locus immediately downstream of
gspB
. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione
S
-transferase-GspB fusion proteins in M99 and in
gly
,
nss
,
gtf
, and
orf4
mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the
gtf
and
orf4
mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in
Escherichia coli
, glycosylation of this protein was observed only in the presence of both
gtf
and
orf4
. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB.
Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector
Authors: Shimada Tomohiro , Makinoshima Hideki , Ogawa Yoshito , Miki Takeyoshi , Maeda Michihisa , Ishihama Akira
Published: October 2004

When
an
Escherichia coli
culture changes from exponential growth to
the stationary phase, expression of growth-related genes levels off,
while a number of stationary-phase-specific genes are turned on. To
gain insight into the growth phase-dependent global regulation of
genome transcription, we analyzed the strength and specificity of
promoters associated with the stationary-phase genes. For the in vivo
assay of promoter activity, 300- to 500-bp DNA fragments upstream from
the translation initiation codon were isolated and inserted into a
newly constructed doubly fluorescent protein (DFP) vector. The activity
of test promoters was determined by measuring the green fluorescent
protein (GFP). To avoid the possible influence of plasmid copy number,
the level of transcription of reference promoter
lac
UV5 on the same plasmid was determined by
measuring the red fluorescent protein (RFP). Thus, the activities of
test promoters could be easily and accurately determined by determining
the GFP/RFP ratio. Analysis of the culture time-dependent variation of
100 test promoters indicated that (i) a major group of the
stationary-phase promoters are up-regulated only in the presence of
RpoS sigma; (ii) the phase-coupled increase in the activity of some
promoters takes place even in the absence of RpoS; and (iii) the
activity of some promoters increases in the absence of RpoS. This
classification was confirmed by testing in vitro transcription by using
reconstituted RpoD and RpoS
holoenzymes.
Isolation of SOS Constitutive Mutants ofEscherichia coli
Authors: O'Reilly Erin K. , Kreuzer Kenneth N.
Published: October 2004

The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen forEscherichia colimutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from adinD1::lacZreporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such aslexA, dam,rep,xerCD,recG, andpolA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, includingdcd, ftsE, ftsX, purF,tdcE, andtynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.
The N Terminus of MinD Contains Determinants Which Affect Its Dynamic Localization and Enzymatic Activity
Authors: Szeto Jason , Acharya Sudeep , Eng Nelson F. , Dillon Jo-Anne R.
Published: October 2004

MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including
Neisseria gonorrhoeae
and
Escherichia coli
. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in
E. coli
. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of
N. gonorrhoeae
MinD (MinD
Ng
) on protein dynamism, localization, and interactions with MinD
Ng
and with MinE
Ng
. Deletions or mutations were generated in the first five residues of MinD
Ng
, and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinD
Ng
interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinD
Ng
mutants could still oscillate from pole to pole in
E. coli
, the GFP-MinD
Ng
oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinD
Ng
proteins lacking the first three residues or with an I5E substitution possessed higher MinE
Ng
-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinD
Ng
are implicated in regulating the enzymatic activity and dynamic localization of the protein.
σ
S
-Dependent Gene Expression at the Onset of Stationary Phase in
Escherichia coli
: Function of σ
S
-Dependent Genes and Identification of Their Promoter Sequences
Authors: Lacour Stephan , Landini Paolo
Published: October 2004

The σ
S
subunit of RNA polymerase, the product of the
rpoS
gene, controls the expression of genes responding to starvation and cellular stresses. Using gene array technology, we investigated
rpoS
-dependent expression at the onset of stationary phase in
Escherichia coli
grown in rich medium. Forty-one genes were expressed at significantly lower levels in an
rpoS
mutant derived from the MG1655 strain; for 10 of these, we also confirmed
rpoS
and stationary-phase dependence by reverse transcription-PCR. Only seven genes (
dps
,
osmE
,
osmY
,
sodC
,
rpsV
,
wrbA
, and
yahO
) had previously been recognized as
rpoS
dependent. Several newly identified
rpoS
-dependent genes are involved in the uptake and metabolism of amino acids, sugars, and iron. Indeed, the
rpoS
mutant strain shows severely impaired growth on some sugars such as fructose and
N
-acetylglucosamine. The
rpoS
gene controls the production of indole, which acts as a signal molecule in stationary-phase cells, via regulation of the
tnaA
-encoded tryptophanase enzyme. Genes involved in protein biosynthesis, encoding the ribosome-associated protein RpsV (
sra
) and the initiation factor IF-1 (
infA
), were also induced in an
rpoS
-dependent fashion. Using primer extension, we determined the promoter sequences of a selection of
rpoS
-regulated genes representative of different functional classes. Significant fractions of these promoters carry sequence features specific for Eσ
S
recognition of the −10 region, such as cytosines at positions −13 (70%) and −12 (30%) as well as a TG motif located upstream of the −10 region (50%), thus supporting the TGN
0-2
C(C/T)ATA(C/A)T consensus sequence recently proposed for σ
S
.
Role of α-Methylacyl Coenzyme A Racemase in the Degradation of Methyl-Branched Alkanes by
Mycobacterium
sp. Strain P101
Authors: Sakai Yasuyoshi , Takahashi Hironori , Wakasa Yuori , Kotani Tetsuya , Yurimoto Hiroya , Miyachi Nobuya , Van Veldhoven Paul P. , Kato Nobuo
Published: October 2004

A new isolate,
Mycobacterium
sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn
5
-derived mutants, we characterized 2 mutants defective in growth on pristane or
n
-hexadecane. A single copy of Tn
5
was found to be inserted into the coding region of
mcr
(α-methylacyl coenzyme A [α-methylacyl-CoA] racemase gene) in mutant P1 and into the coding region of
mls
(malate synthase gene) in mutant H1. Mutant P1 could not grow on methyl-branched alkanes. The recombinant Mcr produced in
Escherichia coli
was confirmed to catalyze racemization of (
R
)-2-methylpentadecanoyl-CoA, with a specific activity of 0.21 μmol · min
−1
 · mg of protein
−1
. Real-time quantitative reverse transcriptase PCR analyses indicated that
mcr
gene expression was enhanced by the methyl-branched alkanes pristane and squalane. Mutant P1 used (
S
)-2-methylbutyric acid for growth but did not use the racemic compound, and growth on
n
-hexadecane was not inhibited by pristane. These results suggested that the oxidation of the methyl-branched alkanoic acid is inhibited by the (
R
) isomer, although the (
R
) isomer was not toxic during growth on
n
-hexadecane. Based on these results, Mcr is suggested to play a critical role in β-oxidation of methyl-branched alkanes in
Mycobacterium
. On the other hand, mutant H1 could not grow on
n
-hexadecane, but it partially retained the ability to grow on pristane. The reduced growth of mutant H1 on pristane suggests that propionyl-CoA is available for cell propagation through the 2-methyl citric acid cycle, since propionyl-CoA is produced through β-oxidation of pristane.
MorA Defines a New Class of Regulators Affecting Flagellar Development and Biofilm Formation in Diverse
Pseudomonas
Species
Authors: Choy Weng-Keong , Zhou Lian , Syn Chris Kiu-Choong , Zhang Lian-Hui , Swarup Sanjay
Published: October 2004

Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in
Pseudomonas putida
. MorA is conserved among diverse
Pseudomonas
species, and homologues are present in all
Pseudomonas
genomes sequenced thus far. In
P. putida
, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In
Pseudomonas aeruginosa
, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of
P. putida
, the motility of the
P. aeruginosa
mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of
Pseudomonas
species.
The
Escherichia coli
DjlA and CbpA Proteins Can Substitute for DnaJ in DnaK-Mediated Protein Disaggregation
Authors: Gur Eyal , Biran Dvora , Shechter Nelia , Genevaux Pierre , Georgopoulos Costa , Ron Eliora Z.
Published: October 2004

The DnaJ (Hsp40) protein of
Escherichia coli
serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other
E. coli
proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in
dnaJ
mutants both CbpA and DjlA are essential for growth at temperatures above 37°C, their in vivo role is poorly understood. Here we show that in a
dnaJ
mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42°C.
ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis
Authors: Tiemann Bernd , Depping Reinhard , Gineikiene Egle , Kaliniene Laura , Nivinskas Rimas , Rüger Wolfgang
Published: October 2004

Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.
The
N
-Acetyl-
d
-Glucosamine Kinase of
Escherichia coli
and Its Role in Murein Recycling
Authors: Uehara Tsuyoshi , Park James T.
Published: October 2004

N
-Acetyl-
d
-glucosamine (GlcNAc) is a major component of bacterial cell wall murein and the lipopolysaccharide of the outer membrane. During growth, over 60% of the murein of the side wall is degraded, and the major products, GlcNAc-anhydro-
N
-acetylmuramyl peptides, are efficiently imported into the cytoplasm and cleaved to release GlcNAc, anhydro-
N
-acetylmuramic acid, murein tripeptide (
l
-Ala-
d
-Glu-
meso
-diaminopimelic acid), and
d
-alanine. Like murein tripeptide, GlcNAc is readily recycled, and this process was thought to involve phosphorylation, since GlcNAc-6-phosphate (GlcNAc-6-P) is efficiently used to synthesize murein or lipopolysaccharide or can be metabolized by glycolysis. Since the gene for GlcNAc kinase had not been identified, in this work we purified GlcNAc kinase (NagK) from
Escherichia coli
cell extracts and identified the gene by determining the N-terminal sequence of the purified kinase. A
nagK
deletion mutant lacked phosphorylated GlcNAc in its cytoplasm, and the cell extract of the mutant did not phosphorylate GlcNAc, indicating that NagK is the only GlcNAc kinase expressed in
E. coli
. Unexpectedly, GlcNAc did not accumulate in a
nagK nagEBACD
mutant, though both GlcNAc and GlcNAc-6-P accumulate in the
nagEBACD
mutant, suggesting the existence of an alternative pathway (presumably repressed by GlcNAc-6-P) that reutilizes GlcNAc without the involvement of NagK.
Role of
hha
and
ler
in Transcriptional Regulation of the
esp
Operon of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Sharma Vijay K. , Zuerner Richard L.
Published: October 2004

The locus of enterocyte effacement (LEE), which includes five major operons (
LEE1
through
LEE4
and
tir
), enables enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 to produce attaching and effacing lesions on host cells. Expression of
LEE2
,
LEE3
, and
tir
is positively regulated by
ler
, a gene located in
LEE1
. Transcriptional regulation of the
esp
operon (
LEE4
), however, is not well defined. Transposon mutagenesis was used to identify transcriptional regulators of the
esp
operon by screening for mutants with increased β-galactosidase activity in an EHEC O157:H7 strain harboring an
esp
::
lac
transcriptional fusion. All mutants with significant increases in β-galactosidase activity had transposon insertions in
hha
(
hha
::Tn). Specific complementation of the
hha
::Tn mutation with a plasmid-encoded copy of
hha
reduced β-galactosidase activity to the level expressed in the parental
esp
::
lac
strain. Purified Hha, however, bound poorly to the
esp
promoter, suggesting that Hha might repress the transcription of a positive regulator of
esp
. Transposon mutagenesis of a Δ
hha esp
::
lac
strain expressing elevated levels of β-galactosidase resulted in
ler
mutants with reduced β-galactosidase activity. Purified Hha bound to the
ler
promoter with a higher affinity, and complementation of a Δ
hha
mutation in a Δ
hha ler
::
lac
strain repressed β-galactosidase activity to the level expressed in a
ler
::
lac
strain. A positive regulatory role of
ler
in
esp
expression was demonstrated by specific binding of Ler to the
esp
promoter, reduced expression of β-galactosidase in Δ
ler esp
::
lac
strains with and without
hha
, and severalfold-increased transcription of
ler
and
espA
in strains lacking
hha
. These results indicate that
hha
-mediated repression of
ler
causes reduced expression of the
esp
operon.
The BfeR Regulator Mediates Enterobactin-Inducible Expression of
Bordetella
Enterobactin Utilization Genes
Authors: Anderson Mark T. , Armstrong Sandra K.
Published: October 2004

Utilization of the enterobactin siderophore by the respiratory pathogens
Bordetella pertussis
and
Bordetella bronchiseptica
is dependent on the BfeA outer membrane receptor. This study determined that production of BfeA was increased significantly in iron-starved bacteria upon supplementation of cultures with enterobactin. A 1.01-kb open reading frame, designated
bfeR
, encoding a predicted positive transcriptional regulator of the AraC family was identified upstream and divergently oriented from
bfeA
. In iron-depleted cultures containing enterobactin, a
Bordetella bfeR
mutant exhibited markedly decreased BfeA receptor production compared to that of the wild-type strain. Additionally,
B. pertussis
and
B. bronchiseptica bfeR
mutants exhibited impaired growth with ferric enterobactin as the sole source of iron, demonstrating that effective enterobactin utilization is
bfeR
dependent. Transcriptional analysis using
bfeA
-
lacZ
reporter fusions in wild-type strains demonstrated that
bfeA
transcription was stimulated in iron-depleted conditions in the presence of enterobactin, compared to modest expression levels in cultures lacking enterobactin. In contrast,
bfeA
transcription in
B. pertussis
and
B. bronchiseptica bfeR
mutants was completely unresponsive to the enterobactin inducer.
bfeA
transcriptional analyses of a
bfeA
mutant demonstrated that induction by enterobactin did not require BfeA receptor-mediated uptake of the siderophore. These studies establish that
bfeR
encodes an enterobactin-dependent positive regulator of
bfeA
transcription in these
Bordetella
species.
Characterization of Nutrient-Induced Dispersion in
Pseudomonas aeruginosa
PAO1 Biofilm
Authors: Sauer K. , Cullen M. C. , Rickard A. H. , Zeef L. A. H. , Davies D. G. , Gilbert P.
Published: October 2004

The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of
Pseudomonas aeruginosa
PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to ∼80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (
fliC
) and correspondingly decreased expression of pilus (
pilA
) genes in dispersed cells. Changes in gene expression associated with dispersion of
P. aeruginosa
biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of
P. aeruginosa
from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of
P. aeruginosa
correlates with a specific dispersal phenotype.
A Nonribosomal Peptide Synthetase with a Novel Domain Organization Is Essential for Siderophore Biosynthesis in
Vibrio anguillarum
Authors: Di Lorenzo Manuela , Poppelaars Sophie , Stork Michiel , Nagasawa Maho , Tolmasky Marcelo E. , Crosa Jorge H.
Published: October 2004

Anguibactin, a siderophore produced by
Vibrio anguillarum
, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. We have identified a gene from the
V. anguillarum
plasmid pJM1 that encodes a 78-kDa NRPS protein termed AngM, which is essential in the biosynthesis of anguibactin. The predicted AngM amino acid sequence shows regions of homology to the consensus sequence for the peptidyl carrier protein (PCP) and the condensation (C) domains of NRPSs, and curiously, these two domains are not associated with an adenylation (A) domain. Substitution by alanine of the serine 215 in the PCP domain and of histidine 406 in the C domain of AngM results in an anguibactin-deficient phenotype, underscoring the importance of these two domains in the function of this protein. The mutations in
angM
that affected anguibactin production also resulted in a dramatic attenuation of the virulence of
V. anguillarum
775, highlighting the importance of this gene in the establishment of a septicemic infection in the vertebrate host. Transcription of the
angM
gene is initiated at an upstream transposase gene promoter that is repressed by the Fur protein in the presence of iron. Analysis of the sequence at this promoter showed that it overlaps the iron transport-biosynthesis promoter and operates in the opposite direction.
The NtcA-Dependent P
1
Promoter Is Utilized for
glnA
Expression in N
2
-Fixing Heterocysts of
Anabaena
sp. Strain PCC 7120
Authors: Valladares Ana , Muro-Pastor Alicia M. , Herrero Antonia , Flores Enrique
Published: October 2004

Expression of the
glnA
gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N
2
-fixing cyanobacterium
Anabaena
sp. strain PCC 7120,
glnA
is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNA
I
originates from a canonical NtcA-dependent promoter (P
1
) and RNA
II
originates from a σ
70
-type promoter (P
2
), RNA
IV
is influenced by NtcA but the corresponding promoter (P
3
) does not have the structure of NtcA-activated promoters. Using RNA isolated from
Anabaena
filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNA
V
, which has previously been detected only in in vitro transcription assays and should originate from P
4
. However, in heterocysts, which are differentiated cells specialized in N
2
fixation, RNA
I
was the almost exclusive
glnA
transcript. Analysis of P

glnA

::
lacZ
fusions containing different fragments of the
glnA
upstream region confirmed that fragments carrying P
1
, P
2
, or P
3
and P
4
have the ability to promote transcription. Mutation of the NtcA-binding site in P
1
eliminated P
1
-directed transcription and allowed increased use of P
2
. The NtcA-binding site in the P
1
promoter and binding of NtcA to this site appear to be key factors in determining
glnA
gene expression in vegetative cells and heterocysts.
Temporal Analysis of
Coxiella burnetii
Morphological Differentiation
Authors: Coleman Sherry A. , Fischer Elizabeth R. , Howe Dale , Mead David J. , Heinzen Robert A.
Published: October 2004

Coxiella burnetii
undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study
C. burnetii
morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of
scvA
, which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of
C. burnetii
is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of
C. burnetii
developmental forms.
AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in
Pseudomonas aeruginosa
Authors: Robles-Price Antonette , Wong Thiang Yian , Sletta Håvard , Valla Svein , Schiller Neal L.
Published: October 2004

Alginate, an exopolysaccharide produced by
Pseudomonas aeruginosa
, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene,
algX
, within the alginate biosynthetic gene cluster on the
P. aeruginosa
chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of
algX
in
P. aeruginosa
strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting
algX
Δ::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The
algX
Δ::Gm mutant was restored to the mucoid phenotype with wild-type
P. aeruginosa algX
provided on a plasmid. The
algX
Δ::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.
Characterization of EvgAS-YdeO-GadE Branched Regulatory Circuit Governing Glutamate-Dependent Acid Resistance inEscherichia coli
Authors: Ma Zhuo , Masuda Nobuhisa , Foster John W.
Published: October 2004

Escherichia coliprefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the fourE. colisystems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, γ-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncoveredevgASandydeOas potential regulators ofgadE, now known to encode the essential activator of thegadAandgadBCgenes. Examination ofevgAandydeOunder normal expression conditions revealed that their products do activategadEexpression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary forgadEexpression in stationary-phase cells grown in complex medium. The response regulator EvgA activatesgadEdirectly and indirectly via induction of the AraC-like regulatorydeO. Evidence obtained usinggadE-lacZoperon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream ofgadE, indicating they all act directly at thegadEpromoter. Since GadE controls the expression of numerous genes besidesgadAandgadBC, the relevance of these regulatory circuits extends beyond acid resistance.
Use of In Vivo Expression Technology To Identify Genes Important in Growth and Survival of
Pseudomonas fluorescens
Pf0-1 in Soil: Discovery of Expressed Sequences with Novel Genetic Organization
Authors: Silby Mark W. , Levy Stuart B.
Published: October 2004

Studies were undertaken to determine the genetic needs for the survival of
Pseudomonas fluorescens
Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches.
Interactions between the PAS and HAMP Domains of theEscherichia coliAerotaxis Receptor Aer
Authors: Watts Kylie J. , Ma Qinhong , Johnson Mark S. , Taylor Barry L.
Published: October 2004

TheEscherichia colienergy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 α-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.
Supercritical sewer flows at a combining junction: A model study of the Edworthy trunk junction, Calgary, Alberta
Authors: Zhao Can-Hua , Zhu David Z , Rajaratnam Nallamuthu
Published: October 2004

An experimental study on supercritical sewer flows at a junction chamber was conducted using a physical model for the Edworthy trunk sewer in Calgary, Alberta. This model study showed that a junction chamber with a small angle of two combining inflows helps to retain kinetic energy of the flow and reduces the water level in the surcharged chamber, thus avoiding the occurrence of fountains. The behavior of the supercritical junction flow in the chamber was investigated. Transition of flow regimes in the chamber was studied and three flow regimes were identified: regime I features shock waves in open-channel flow in the chamber; regime II is characterized by surcharge flow in the chamber and orifice flow in the outlet pipe; and regime III is associated with pipe-full flow in the outlet pipe following the surcharged chamber. It was found that regime II could be the most critical condition for the consideration of water level in a surcharged chamber. A theoretical analysis of regime II flow was carried out, and the degree of submergence in the chamber can be satisfactorily predicted for given inflow conditions. The effects of a rounded entrance of the outlet pipe and the benching in the junction chamber were also evaluated experimentally. Key words: sewer hydraulics, supercritical flow, energy loss, junction manhole.
Large-scale flow structures and stability in shallow flows
Authors: Socolofsky Scott A , Jirka Gerhard H
Published: October 2004

Shallow turbulent flows occurring in wide rivers, estuaries, lakes or coastal regions, as well as the atmosphere, are readily susceptible to transverse disturbances that lead to two-dimensional coherent structures. The shallow jet, the shallow wake, and the shallow mixing layer are examples of such flow patterns. A linear stability analysis incorporating the effects of bottom friction and viscosity is applied to these flows to determine the criteria for absolute and convective instability and for stabilization of the flow. All three flow types exhibit instabilities within the range of natural flows. Three mechanisms for large-scale instability generation, namely topographic forcing, transverse velocity shear, and secondary instabilities of the base flow, are compared to the stability calculations. Based on the results, topographic forcing, and to a lesser extent, transverse shear are expected to have both absolute and convective instabilities; whereas, secondary instabilities of the base flow are expected to generate primarily convective instabilities. The resulting large-scale coherent structures greatly influence the mixing and transport of pollutants and momentum that are released into such flows. Key words: shallow flow, shallow jet, shallow wake, shallow mixing layer, linear stability analysis, bottom friction, Chebyshev polynomial method.