bluuebunny/crossref_metadata_2025 huggingface dataset:
Function of the
cypX
and
moxY
Genes in Aflatoxin Biosynthesis in
Aspergillus parasiticus
Authors: Wen Ying , Hatabayashi Hidemi , Arai Hatsue , Kitamoto Hiroko K. , Yabe Kimiko
Published: June 2005

The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the
cypX
and
moxY
genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic
Aspergillus parasiticus
NRRL 2999. The
cypX
-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The
moxY
-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that
cypX
encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and
moxY
encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.
The HtrA Protease of
Campylobacter jejuni
Is Required for Heat and Oxygen Tolerance and for Optimal Interaction with Human Epithelial Cells
Authors: Brøndsted Lone , Andersen Marianne Thorup , Parker Mary , Jørgensen Kirsten , Ingmer Hanne
Published: June 2005

Campylobacter jejuni
is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in
C. jejuni
stress tolerance. A
C. jejuni htrA
mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44°C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the
htrA
mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the
htrA
mutant, suggesting that accumulation of nonnative proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of
C. jejuni
, the
htrA
mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H
2
O
2
. Using tissue cultures of human epithelial cells (INT407), we found that the
htrA
mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the
htrA
mutant. Thus, our results suggest that in
C. jejuni
, HtrA is important for growth during stressful conditions and has an impact on virulence.
Effects of Deregulation of Methionine Biosynthesis on Methionine Excretion in
Escherichia coli
Authors: Usuda Yoshihiro , Kurahashi Osamu
Published: June 2005

Several regulators of methionine biosynthesis have been reported in
Escherichia coli
, which might represent barriers to the production of excess
l
-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (
metJ
) and threonine biosynthetic (
thrBC
) genes were introduced into the W3110 wild-type strain of
E. coli
. Mutations of the
metK
gene encoding
S
-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the
metK
structural gene were then introduced into
metJ
and
thrBC
double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the
metA
gene encoding homoserine succinyltransferase were detected in α-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a
14
C-labeled homoserine assay. Three
metA
mutations were introduced, using expression plasmids, into an
E. coli
strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.
Inhibition of Efflux Transporter-Mediated Fungicide Resistance in
Pyrenophora tritici-repentis
by a Derivative of 4′-Hydroxyflavone and Enhancement of Fungicide Activity
Authors: Reimann Sven , Deising Holger B.
Published: June 2005

Populations of the causal agent of wheat tan spot,
Pyrenophora tritici-repentis
, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C
14
-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in
P. tritici-repentis
. Application of this compound in combination with fungicides shifted fungicide-resistant
P. tritici-repentis
isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.
Physiological Characterization of the
ARO10
-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of
Saccharomyces cerevisiae
Authors: Vuralhan Zeynep , Luttik Marijke A. H. , Tai Siew Leng , Boer Viktor M. , Morais Marcos A. , Schipper Dick , Almering Marinka J. H. , Kötter Peter , Dickinson J. Richard , Daran Jean-Marc , Pronk Jack T.
Published: June 2005

Aerobic, glucose-limited chemostat cultures of
Saccharomyces cerevisiae
CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (
PDC1
,
PDC5
,
PDC6
,
ARO10
, and
THI3
), only
ARO10
was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of
ARO10
in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of
ARO10
in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when
ARO10
was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the
ARO10
-dependent, broad-substrate-specificity decarboxylase activity.
Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from
Bacillus megaterium
IWG3 on Stabilization of Its Oligomeric State
Authors: Baik Sang-Ho , Michel Fabrice , Aghajari Nushin , Haser Richard , Harayama Shigeaki
Published: June 2005

A thermostable glucose dehydrogenase (GlcDH) mutant of
Bacillus megaterium
IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem.
264:
6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol.
61:
329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.
Evolutionary Relationships of Three New Species of
Enterobacteriaceae
Living as Symbionts of Aphids and Other Insects
Authors: Moran Nancy A. , Russell Jacob A. , Koga Ryuichi , Fukatsu Takema
Published: June 2005

Ecological studies on three bacterial lineages symbiotic in aphids have shown that they impose a variety of effects on their hosts, including resistance to parasitoids and tolerance to heat stress. Phylogenetic analyses of partial sequences of
gyrB
and
recA
are consistent with previous analyses limited to 16S rRNA gene sequences and yield improved confidence of the evolutionary relationships of these symbionts. All three symbionts are in the
Enterobacteriaceae
. One of the symbionts, here given the provisional designation “
Candidatus
Serratia symbiotica,” is a
Serratia
species that has acquired a symbiotic lifestyle. The other two symbionts, here designated “
Candidatus
Hamiltonella defensa” and “
Candidatus
Regiella insecticola,” are sister groups to one another and together show a relationship to species of
Photorhabdus
.
Complete Genome Sequence of φHSIC, a Pseudotemperate Marine Phage of
Listonella pelagia
Authors: Paul John H. , Williamson Shannon J. , Long Amy , Authement R. Nathan , John David , Segall Anca M. , Rohwer Forest L. , Androlewicz Matthew , Patterson Stacey
Published: June 2005

The genome for the marine pseudotemperate member of the
Siphoviridae
φHSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. φHSIC enters into a pseudolysogenic relationship with its host,
Listonella pelagia
, characterized by sigmoidal growth curves producing >10
9
cells/ml and >10
11
phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a β subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of
Salmonella
phage P22 and
Shigella
phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), φHSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.
Involvement of Bacterial Quorum-Sensing Signals in Spoilage of Bean Sprouts
Authors: Rasch Maria , Andersen Jens Bo , Nielsen Kristian Fog , Flodgaard Lars Ravn , Christensen Henrik , Givskov Michael , Gram Lone
Published: June 2005

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10
8
to 10
9
CFU/g. AHL-producing bacteria (
Enterobacteriaceae
and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts. Thin-layer chromatography and liquid chromatography-high-resolution mass spectrometry revealed the presence of
N
-3-oxo-hexanoyl-
l
-homoserine lactone in spoiled bean sprouts and in extracts from pure cultures of bacteria. During normal spoilage, the pH of the sprouts increased due to proteolytic activity, and the higher pH probably facilitated the activity of pectate lyase. The AHL synthetase gene (
I
gene) from a spoilage
Pectobacterium
was cloned, sequenced, and inactivated in the parent strain. The predicted amino acid sequence showed 97% homology to HslI and CarI in
Erwinia carotovora
. Spoilage of laboratory bean sprouts inoculated with the AHL-negative mutant was delayed compared to sprouts inoculated with the wild type, and the AHL-negative mutant did not cause the pH to rise. Compared to the wild-type strain, the AHL-negative mutant had significantly reduced protease and pectinase activities and was negative in an iron chelation (siderophore) assay. This is the first study demonstrating AHL regulation of iron chelation in
Enterobacteriaceae
. The present study clearly demonstrates that the bacterial spoilage of some food products is influenced by quorum-sensing-regulated phenotypes, and understanding these processes may be useful in the development of novel food preservation additives that specifically block the quorum-sensing systems.
Development and Evaluation of a Broadly Reactive TaqMan Assay for Rapid Detection of Hepatitis A Virus
Authors: Jothikumar N. , Cromeans T. L. , Sobsey M. D. , Robertson B. H.
Published: June 2005

Primers and a TaqMan probe for the 5′-untranslated region (UTR) of the hepatitis A virus (HAV) genome were designed and evaluated. The assay detected 0.5 infectious units of HAV and 40 copies of a synthetic transcript and provides an important screening tool for rapid quantitative HAV detection in clinical or environmental samples.
Evidence for the Presence of an Alternative Glucose Transport System in
Clostridium beijerinckii
NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101
Authors: Lee Jieun , Mitchell Wilfrid J. , Tangney Martin , Blaschek H. P.
Published: June 2005

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by
Clostridium beijerinckii
provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type
C. beijerinckii
NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.
Deletion of the
datA
Site Does Not Affect Once-per-Cell-Cycle Timing but Induces Rifampin-Resistant Replication
Authors: Morigen  , Molina Felipe , Skarstad Kirsten
Published: June 2005

In
Escherichia coli
, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the
datA
site. The
datA
site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in Δ
datA
cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the
datA
site is not required to prevent reinitiation and limit initiation of replication to once per generation. The
datA
site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.
Complete Nucleotide Sequence of the LE1 Prophage from the SpirocheteLeptospira biflexaand Characterization of Its Replication and Partition Functions
Authors: Bourhy Pascale , Frangeul Lionel , Couvé Elisabeth , Glaser Philippe , Saint Girons Isabelle , Picardeau Mathieu
Published: June 2005

The first and, to date, only extrachromosomal circular replicon identified in the spirocheteLeptospirais the LE1 prophage fromLeptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content ofLeptospiraspp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or inL. biflexalate after infection. Since the LE1 prophage replicates autonomously as a circular replicon inL. biflexa, we were able to engineer anL. biflexa-Escherichia colishuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol.182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Θ-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of arepencoding a protein of 180 amino acids. Upstream fromrep, putative partitioning genes, calledparAandparB, were found to be similar to theparloci inBorreliaplasmids. A significant increase of plasmid stability inL. biflexacan be seen only when bothparAandparBare present. These results enable the construction of new shuttle vectors for studying the genetics ofLeptospiraspp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.
Temperature-Dependent Conjugative Transfer of R27: Role of Chromosome- and Plasmid-Encoded Hha and H-NS Proteins
Authors: Forns Núria , Baños Rosa C. , Balsalobre Carlos , Juárez Antonio , Madrid Cristina
Published: June 2005

IncHI plasmids encode multiple-antibiotic resistance inSalmonella entericaserovar Typhi. These plasmids have been considered to play a relevant role in the persistence and reemergence of this microorganism. The IncHI1 plasmid R27, which can be considered the prototype of IncHI plasmids, is thermosensitive for transfer. Conjugation frequency is highest at low temperature (25 to 30°C), decreasing when temperature increases. R27 codifies an H-NS-like protein (open reading frame 164 [ORF164]) and an Hha-like protein (ORF182). The H-NS and Hha proteins participate in the thermoregulation of gene expression inEscherichia coli. Here we investigated the hypothetical role of such proteins in thermoregulation of R27 conjugation. At a nonpermissive temperature (33°C), transcription of several ORFs in both transfer region 1 (Tra1) and Tra2 from R27 is upregulated in cells depleted of Hha-like and H-NS-like proteins. Both chromosome- and plasmid-encoded Hha and H-NS proteins appear to potentially modulate R27 transfer. The function of R27-encoded Hha-like and H-NS proteins is not restricted to modulation of R27 transfer. Different mutant phenotypes associated with both chromosomalhhaandhnsmutations are compensated in cells harboring R27.
Transcriptional Regulation of the Flavohemoglobin Gene for Aerobic Nitric Oxide Detoxification by the Second Nitric Oxide-Responsive Regulator of
Pseudomonas aeruginosa
Authors: Arai Hiroyuki , Hayashi Michiko , Kuroi Azusa , Ishii Masaharu , Igarashi Yasuo
Published: June 2005

The regulatory gene for a σ
54
-dependent-type transcriptional regulator,
fhpR
, is located upstream of the
fhp
gene for flavohemoglobin in
Pseudomonas aeruginosa
. Transcription of
fhp
was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the
fhp
promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for σ
54
was found in the promoter region of
fhp
, but an FNR (fumarate nitrate reductase regulator) box was not. The
fhp
promoter was inactive in the
fhpR
or
rpoN
mutant strain, suggesting that the NO-sensing regulation of the
fhp
promoter was mediated by FhpR. The DNR-dependent denitrification promoters (
nirS
,
norC
, and
nosR
) were active in the
fhpR
or
rpoN
mutants. These results indicated that
P. aeruginosa
has at least two independent NO-responsive regulatory systems. The
fhp
or
fhpR
mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in
P. aeruginosa
is detoxification of NO under aerobic conditions.
Pneumococcal HtrA Protease Mediates Inhibition of Competence by the CiaRH Two-Component Signaling System
Authors: Sebert M. E. , Patel K. P. , Plotnick M. , Weiser J. N.
Published: June 2005

Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation inStreptococcus pneumoniaethrough a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression ofhtrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of aciaRH+strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.
Regulation of Bacteriocin Production in
Streptococcus mutans
by the Quorum-Sensing System Required for Development of Genetic Competence
Authors: van der Ploeg Jan R.
Published: June 2005

In
Streptococcus mutans
, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by
comC
). Here, it is shown that the same system is also required for expression of the
nlmAB
genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional
nlmAB
′-
lacZ
fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the
comDE
genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the
nlmAB
promoter region led to a decrease in CSP-dependent expression of
nlmAB
′
-lacZ
. In agreement with these results, a
comDE
mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.
Membrane-Associated Maturation of the Heterotetrameric Nitrate Reductase ofThermus thermophilus
Authors: Zafra Olga , Cava Felipe , Blasco Francis , Magalon Axel , Berenguer Jose
Published: June 2005

Thenaroperon, coding for the respiratory nitrate reductase ofThermus thermophilus(NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants ofT. thermophiluslacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized bynarJmutants, but in contrast to itsEscherichia colihomologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that ofE. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.
Characterization of the Small Untranslated RNA RyhB and Its Regulon inVibrio cholerae
Authors: Davis Brigid M. , Quinones Mariam , Pratt Jason , Ding Yanpeng , Waldor Matthew K.
Published: June 2005

Numerous small untranslated RNAs (sRNAs) have been identified inEscherichia coliin recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved inVibrio cholerae, and both identification and characterization of sRNAs inV. choleraeremain at a preliminary stage. We have characterized one of the few sRNAs conserved betweenE. coliandV. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB inV. choleraeis significantly larger than inE. coli. As inE. coli,V. choleraeRyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB inV. choleraeandE. coliappear to differ, although some overlap is evident. Analysis of gene expression inV. choleraein the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host byV. cholerae.
A VicRK Signal Transduction System in
Streptococcus mutans
Affects
gtfBCD
,
gbpB
, and
ftf
Expression, Biofilm Formation, and Genetic Competence Development
Authors: Senadheera M. Dilani , Guggenheim Bernard , Spatafora Grace A. , Huang Yi-Chen Cathy , Choi Jison , Hung David C. I. , Treglown Jennifer S. , Goodman Steven D. , Ellen Richard P. , Cvitkovitch Dennis G.
Published: June 2005

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the
vicRK
signal transduction system in
Streptococcus mutans
. A
vicK
(putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a
vicR
(putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the
vicRKX
operon (Smuvic
+
). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the
vicK
mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic
+
. Based on real-time PCR, Smuvic
+
showed increased
gtfBCD
,
gbpB
, and
ftf
expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the
gtfB
,
gtfC
, and
ftf
genes. Also, transformation efficiency in the presence or absence of the
S. mutans
competence-stimulating peptide was altered for the
vic
mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of
vicK
did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the
vicRK
signal transduction system in regulating several important physiological processes in
S. mutans
.
ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in EnterohemorrhagicEscherichia coli
Authors: Iyoda Sunao , Watanabe Haruo
Published: June 2005

Expression of the type III protein secretion system (TTSS), encoded in the locus of enterocyte effacement (LEE) of enterohemorrhagicEscherichia coli(EHEC), has been shown to be controlled by various regulators. In a search for additional regulatory genes, we identified a DNA fragment containingclpXandclpPthat has a positive regulatory effect on LEE expression in EHEC O157. The expression of LEE-encoded Esp proteins was significantly reduced in aclpXPdeletion mutant. Deletion ofgrlR, a negative regulatory gene within LEE, markedly increased LEE expression even in theclpXPmutant. To verify the regulatory mechanism of GrlR expression, a chromosomal epitope-tagged allele ofgrlR(grlR-FLAG) was constructed. GrlR-FLAG expression was increased significantly in theclpXPdeletion mutant, suggesting that the GrlR level is under the control of ClpXP, and this regulation is critical for the ClpXP-dependent expression of LEE in EHEC. Deletion ofrpoS, the gene encoding a stationary-phase-inducing sigma factor that is a substrate for ClpXP protease, partially restored LEE expression in theclpXPmutant. A multicopy plasmid carryingrpoSstrongly repressed expression of Esp proteins, suggesting that positive regulation by ClpXP is partially mediated through a negative effect of RpoS on LEE expression. We also found thatrpoSdeletion induces transcription ofpchA, which encodes one of the positive regulators for LEE expression in EHEC. These results suggest that ClpXP controls expression of LEE through the regulation of RpoS and GrlR levels in EHEC.
Genomic Sequence and Receptor for the
Vibrio cholerae
Phage KSF-1Φ: Evolutionary Divergence among Filamentous Vibriophages Mediating Lateral Gene Transfer
Authors: Faruque Shah M. , Bin Naser Iftekhar , Fujihara Kazutaka , Diraphat Pornphan , Chowdhury Nityananda , Kamruzzaman M. , Qadri Firdausi , Yamasaki Shinji , Ghosh A. N. , Mekalanos John J.
Published: June 2005

KSF-1Φ, a novel filamentous phage of
Vibrio cholerae
, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1Φ infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the
mshA
gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1Φ infection, naturally occurring MSHA-negative strains and an
mshA
deletion mutant were resistant. Furthermore,
d
-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1Φ. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1Φ, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXΦ, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of
V. cholerae
strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to
V. cholerae
evolution.
Characterization of Gla
KP
, a UDP-Galacturonic Acid C4-Epimerase from
Klebsiella pneumoniae
with Extended Substrate Specificity
Authors: Frirdich Emilisa , Whitfield Chris
Published: June 2005

In
Escherichia coli
and
Salmonella enterica
, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast,
Klebsiella pneumoniae
lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. Gla
KP
is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of Gla
KP
, and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the

K
m

for UDP-glucuronic acid of 13.0 μM. Gla
KP
exists as a dimer in its native form. NAD
+
/NADH is tightly bound by the enzyme and addition of supplementary NAD
+
is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. Gla
KP
was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-
N
-acetylglucosamine/UDP-
N
-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded
gla
KP
partially complemented a
galE
mutation in
S. enterica
and in
K. pneumoniae
; however, chromosomal
gla
KP
could not substitute for
galE
in a
K. pneumoniae galE
mutant in vivo.
Two Thimet Oligopeptidase-Like Pz Peptidases Produced by a Collagen- Degrading Thermophile,
Geobacillus collagenovorans
MO-1
Authors: Miyake Ryoma , Shigeri Yasushi , Tatsu Yoshiro , Yumoto Noboru , Umekawa Midori , Tsujimoto Yoshiyuki , Matsui Hiroshi , Watanabe Kunihiko
Published: June 2005

A collagen-degrading thermophile,
Geobacillus collagenovorans
MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-
d
-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in
G. collagenovorans
MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.
Identification of the Ω4406 Regulatory Region, a Developmental Promoter of
Myxococcus xanthus
, and a DNA Segment Responsible for Chromosomal Position-Dependent Inhibition of Gene Expression
Authors: Loconto Jennifer , Viswanathan Poorna , Nowak Scott J. , Gloudemans Monica , Kroos Lee
Published: June 2005

When starved,
Myxococcus xanthus
cells send signals to each other that coordinate their movements, gene expression, and differentiation. C-signaling requires cell-cell contact, and increasing contact brought about by cell alignment in aggregates is thought to increase C-signaling, which induces expression of many genes, causing rod-shaped cells to differentiate into spherical spores. C-signaling involves the product of the
csgA
gene. A
csgA
mutant fails to express many genes that are normally induced after about 6 h into the developmental process. One such gene was identified by insertion of Tn
5 lac
at site Ω4406 in the
M. xanthus
chromosome. Tn
5 lac
fused transcription of
lacZ
to the upstream Ω4406 promoter. In this study, the Ω4406 promoter region was identified by analyzing mRNA and by testing different upstream DNA segments for the ability to drive developmental
lacZ
expression in
M. xanthus
. The 5′ end of Ω4406 mRNA mapped to approximately 1.3 kb upstream of the Tn
5 lac
insertion. A 1.0-kb DNA segment from 0.8 to 1.8 kb upstream of the Tn
5 lac
insertion, when fused to
lacZ
and integrated at a phage attachment site in the
M. xanthus
chromosome, showed a similar pattern of developmental expression as Tn
5 lac
Ω4406. The DNA sequence upstream of the putative transcriptional start site was strikingly similar to promoter regions of other C-signal-dependent genes. Developmental
lacZ
expression from the 1.0-kb segment was abolished in a
csgA
mutant but was restored upon codevelopment of the
csgA
mutant with wild-type cells, which supply C-signal, demonstrating that the Ω4406 promoter responds to extracellular C-signaling. Interestingly, the 0.8-kb DNA segment immediately upstream of Tn
5 lac
Ω4406 inhibited expression of a downstream
lacZ
reporter in transcriptional fusions integrated at a phage attachment site in the chromosome but not at the normal Ω4406 location. To our knowledge, this is the first example in
M. xanthus
of a chromosomal position-dependent effect on gene expression attributable to a DNA segment outside the promoter region.
Evolution of Transcription Regulatory Genes Is Linked to Niche Specialization in the Bacterial Pathogen
Streptococcus pyogenes
Authors: Bessen Debra E. , Manoharan Anand , Luo Feng , Wertz John E. , Robinson D. Ashley
Published: June 2005

Streptococcus pyogenes
is a highly prevalent bacterial pathogen, most often giving rise to superficial infections at the throat or skin of its human host. Three genotype-defined subpopulations of strains exhibiting strong tropisms for either the throat or skin (specialists) or having no obvious tissue site preference (generalists) are recognized. Since the microenvironments at the throat and skin are distinct, the signal transduction pathways leading to the control of gene expression may also differ for throat versus skin strains of
S. pyogenes
. Two loci (
mga
and
rofA/nra
) encoding global regulators of virulence gene expression are positioned 300 kb apart on the genome; each contains alleles forming two major sequence clusters of ∼25 to 30% divergence that are under balancing selection. Strong linkage disequilibrium is observed between sequence clusters of the transcription regulatory loci and the subpopulations of throat and skin specialists, against a background of high recombination rates among housekeeping genes. A taxonomically distinct commensal species (
Streptococcus dysgalactiae
subspecies
equisimilus
) shares highly homologous
rof
alleles. The findings provide strong support for a mechanism underlying niche specialization that involves orthologous replacement of regulatory genes following interspecies horizontal transfer, although the directionality of gene exchange remains unknown.
Requirement of themymAOperon for Appropriate Cell Wall Ultrastructure and Persistence ofMycobacterium tuberculosisin the Spleens of Guinea Pigs
Authors: Singh Amit , Gupta Radhika , Vishwakarma R. A. , Narayanan P. R. , Paramasivan C. N. , Ramanathan V. D. , Tyagi Anil K.
Published: June 2005

We had recently reported that themymAoperon (Rv3083 to Rv3089) ofMycobacterium tuberculosisis regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope ofM. tuberculosis. In this study, we further show that avirSmutant (MtbΔvirS) and amymAmutant (Mtbmym::hyg) ofM. tuberculosisexhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C24and C26fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption ofvirSandmymAgenes impairs the ability ofM. tuberculosisto survive in activated macrophages, but not in resting macrophages, suggesting the importance of themymAoperon in protecting the bacterium against harsher conditions. Infection of guinea pigs with MtbΔvirS, Mtbmym::hyg, and the parental strain resulted in an ∼800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of thevirSgene into MtbΔvirS. These observations show the important role of themymAoperon in the pathogenesis ofM. tuberculosisat later stages of the disease.
Structural Characterization and Assembly of the Distal Tail Structure of the Temperate Lactococcal Bacteriophage TP901-1
Authors: Vegge Christina S. , Brøndsted Lone , Neve Horst , Mc Grath Stephen , van Sinderen Douwe , Vogensen Finn K.
Published: June 2005

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate
Lactococcus lactis
phage TP901-1 of the
Siphoviridae
family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes
dit
(
orf46
),

tal
TP901-1

(
orf47
),
bppU
(
orf48
),
bppL
(
orf49
), and
orf50
. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal
TP901-1
were shown to be structural tail proteins essential for tail formation, and Tal
TP901-1
was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.