bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of
Campylobacter jejuni
Proteins Recognized by Maternal Antibodies of Chickens
Authors: Shoaf-Sweeney Kari D. , Larson Charles L. , Tang Xiaoting , Konkel Michael E.
Published: September 2008

Campylobacter jejuni
is one of the leading bacterial causes of food-borne gastroenteritis. Infection with
C. jejuni
is frequently acquired through the consumption of undercooked poultry or foods cross-contaminated with raw poultry. Given the importance of poultry as a reservoir for
Campylobacter
organisms, investigators have performed studies to understand the protective role of maternal antibodies in the ecology of
Campylobacter
colonization of poultry. In a previous study, chicks with maternal antibodies generated against the S3B strain of
C. jejuni
provided protection against
Campylobacter
colonization (O. Sahin, N. Luo, S. Huang, and Q. Zhang, Appl. Environ. Microbiol. 69:5372-5379, 2003). We obtained serum samples, collectively referred to as the
C. jejuni
S3B-SPF sera, from the previous study. These sera were determined to contain maternal antibodies that reacted against
C. jejuni
whole-cell lysates as judged by enzyme-linked immunosorbent assay. The antigens recognized by the
C. jejuni
S3B-SPF antibodies were identified by immunoblot analysis, coupled with mass spectrometry, of
C. jejuni
outer membrane protein extracts. This approach led to the identification of
C. jejuni
proteins recognized by the maternal antibodies, including the flagellin proteins and CadF adhesin. In vitro assays revealed that the
C. jejuni
S3B-SPF sera retarded the motility of the
C. jejuni
S3B homologous strain but did not retard the motility of a heterologous strain of
C. jejuni
(81-176). This finding provides a possible mechanism explaining why maternal antibodies confer enhanced protection against challenge with a homologous strain compared to a heterologous strain. Collectively, this study provides a list of
C. jejuni
proteins against which protective antibodies are generated in hens and passed to chicks.
Estimating Single-Cell Lag Times via a Bayesian Scheme
Authors: Malakar P. K. , Barker G. C.
Published: September 2008

Network models offer computationally efficient tools for estimating the variability of single-cell lag phases. Currently, optical methods for estimating the variability of single-cell lag phases use single-cell inocula and are technically challenging. A Bayesian network model incorporating small uncertain inocula addresses these limitations.
Global Transcription and Metabolic Flux Analysis ofEscherichia coliin Glucose-Limited Fed-Batch Cultivations
Authors: Lemuth K. , Hardiman T. , Winter S. , Pfeiffer D. , Keller M. A. , Lange S. , Reuss M. , Schmid R. D. , Siemann-Herzberg M.
Published: September 2008

A time series of whole-genome transcription profiling ofEscherichia coliK-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic “work mode.” σS-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of theE. colicentral carbon metabolism through thecrp,cra, andrelA/spoTmodulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.
Effective Mutagenesis of
Vibrio fischeri
by Using Hyperactive Mini-Tn
5
Derivatives
Authors: Lyell Noreen L. , Dunn Anne K. , Bose Jeffrey L. , Vescovi Susan L. , Stabb Eric V.
Published: September 2008

We have developed a transposon mutagenesis system for
Vibrio fischeri
ES114 that utilizes a hyperactive mutant Tn
5
transposase (E54K and M56A) and optimized transposon ends. Using a conjugation-based procedure, we obtained independent single-insertion mini-Tn
5
mutants at a rate of ∼10
−6
. This simple and inexpensive technique represents a significant improvement over previous methods for transposon mutagenesis of
V. fischeri
and should be applicable to many other bacteria.
Frequent Occurrence of Human-Associated Microsporidia in Fecal Droppings of Urban Pigeons in Amsterdam, The Netherlands
Authors: Bart Aldert , Wentink-Bonnema Ellen M. , Heddema Edou R. , Buijs Jan , van Gool Tom
Published: September 2008

Human-associated microsporidia were frequently observed in fecal samples of 331 feral pigeons in Amsterdam, The Netherlands, obtained during high- and low-breeding periods. Thirty-six of 331 samples (11%) contained the human pathogens
Enterocytozoon bieneusi
(
n
= 18),
Encephalitozoon hellem
(
n
= 11),
Encephalitozoon cuniculi
(
n
= 6), and
Encephalitozoon intestinalis
(
n
= 1); 5 samples contained other microsporidia. Pigeon feces can be an important source of human microsporidian infection.
High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered
Escherichia coli
Strain
Authors: Zhu Yihui , Eiteman Mark A. , Altman Ronni , Altman Elliot
Published: September 2008

We report pyruvate formation in
Escherichia coli
strain ALS929 containing mutations in the
aceEF
,
pfl
,
poxB
,
pps
, and
ldhA
genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h
−1
. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in
atpFH
and
arcA
(strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h
−1
achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.
Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene
RAG1
in
Kluyveromyces lactis
Authors: Bao Wei-Guo , Guiard Bernard , Fang Zi-An , Donnini Claudia , Gervais Michel , Passos Flavia M. Lopes , Ferrero Iliana , Fukuhara Hiroshi , Bolotin-Fukuhara Monique
Published: September 2008

The
HAP1
(
CYP1
) gene product of
Saccharomyces cerevisiae
is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative
S. cerevisiae
,
Kluyveromyces lactis
is an aerobic yeast species which shows different oxygen responses. We examined the role of the
HAP1
-equivalent gene (Kl
HAP1
) in
K. lactis
. KlHap1p showed a number of sequence features and some gene targets (such as Kl
CYC1
) in common with its
S. cerevisiae
counterpart, and Kl
HAP1
was capable of complementing the
hap1
mutation. However, the Kl
HAP1
disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene
RAG1
and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKl
hap1
mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of
K. lactis
.
ESCRT-III Protein Snf7 Mediates High-Level Expression of the
SUC2
Gene via the Rim101 Pathway
Authors: Weiß Peter , Huppert Stefanie , Kölling Ralf
Published: September 2008

The yeast (
Saccharomyces cerevisiae
) Snf7 family consists of six highly charged, coiled-coil-forming proteins involved in multivesicular body (MVB) formation. Although all proteins perform a common function at late endosomes, individual mutants also show distinct phenotypes. This suggests that Snf7 homologues have additional functions separate from their role in MVB formation. In this report, we explored the molecular basis for the
s
ucrose-
n
on
f
ermenting phenotype of
snf7
mutants. Our Northern blotting experiments provide evidence that Snf7 is involved in the regulation of
SUC2
transcription. The Snf7-dependent regulation of
SUC2
transcription does not appear to involve the transcription factor Mig1, since Mig1 phosphorylation after glucose derepression was not affected in a Δs
nf7
mutant. Instead, we show that Snf7 influences
SUC2
expression by regulating the level of the transcription factor Nrg1. Snf7 exerts its effects on Nrg1 levels through activation of the transcription factor Rim101, which is part of the yeast alkaline response pathway (“Rim101 pathway”). This is supported by the findings that deletion of
RIM101
or overexpression of
NRG1
from the
ADH1
promoter leads to the same
SUC2
expression level as deletion of
SNF7
. In addition, deletion of other components of the Rim101 pathway, like
RIM13
and
RIM20
, led to the same growth phenotype on raffinose media as deletion of
SNF7
. Furthermore, Snf7 turned out to be dispensable for
SUC2
expression in an
NRG1
deletion background. Thus, the effects of Snf7 on
SUC2
expression can be completely accounted for by its effect on Nrg1 levels.
The Synthetic Genetic Network around
PKC1
Identifies Novel Modulators and Components of Protein Kinase C Signaling in
Saccharomyces cerevisiae
Authors: Krause Sue A. , Xu Hong , Gray Joseph V.
Published: September 2008

Budding yeast
Saccharomyces cerevisiae
contains one protein kinase C (PKC) isozyme encoded by the essential gene
PKC1
. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around
PKC1
by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around
PKC1
directly and efficiently identifies known and novel components of PKC signaling in yeast.
The Cell Wall of the Human Pathogen
Candida glabrata
: Differential Incorporation of Novel Adhesin-Like Wall Proteins
Authors: de Groot Piet W. J. , Kraneveld Eefje A. , Yin Qing Yuan , Dekker Henk L. , Groß Uwe , Crielaard Wim , de Koster Chris G. , Bader Oliver , Klis Frans M. , Weig Michael
Published: September 2008

The cell wall of the human pathogen
Candida glabrata
governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of
C. glabrata
using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that
C. glabrata
walls incorporate 50% more protein than
Saccharomyces cerevisiae
walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that
C. glabrata
walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-β-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-β-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi,
C. glabrata
contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like
EPA
family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of
C. glabrata
contributes to its adaptability and virulence.
Nonapoptotic Death of
Saccharomyces cerevisiae
Cells That Is Stimulated by Hsp90 and Inhibited by Calcineurin and Cmk2 in Response to Endoplasmic Reticulum Stresses
Authors: Dudgeon Drew D. , Zhang Nannan , Ositelu Olufisayo O. , Kim Hyemin , Cunningham Kyle W.
Published: September 2008

Endoplasmic reticulum (ER) stress can trigger apoptosis and necrosis in many types of mammalian cells. Previous studies in yeast found little or no cell death in response to the ER stressor tunicamycin, but a recent study suggested widespread apoptosis-like death. Here we show that wild-type laboratory
Saccharomyces cerevisiae
cells responding to tunicamycin die by nonapoptotic mechanisms in low-osmolyte culture media and survive for long periods of time in standard synthetic media. Survival requires calcineurin, a Ca
2+
/calmodulin-dependent protein phosphatase, but none of its known targets. The Ca
2+
/calmodulin-dependent protein kinase Cmk2 was identified as an indirect target of calcineurin that suppresses death of calcineurin-deficient cells. Death of Cmk2- and/or calcineurin-deficient
S. cerevisiae
cells was preceded by accumulation of reactive oxygen species but was not associated with hallmarks of apoptosis and was not dependent on Mca1, Aif1, Nuc1, or other factors implicated in apoptosis-like death. Cmk2 and calcineurin also independently suppressed the death of
S. cerevisiae
cells responding to dithiothreitol or miconazole, a common azole-class antifungal drug. Though inhibitors of Hsp90 have been shown to diminish calcineurin signaling in
S. cerevisiae
and to synergistically inhibit growth in combination with azoles, they did not stimulate death of
S. cerevisiae
cells in combination with miconazole or tunicamycin, and instead they prevented the death of calcineurin- and Cmk2-deficient cells. These findings reveal a novel prodeath role for Hsp90 and antideath roles for calcineurin and Cmk2 that extend the life span of
S. cerevisiae
cells responding to both natural and clinical antifungal compounds.
SigF Controls Carotenoid Pigment Production and Affects Transformation Efficiency and Hydrogen Peroxide Sensitivity in
Mycobacterium smegmatis
Authors: Provvedi Roberta , Kocíncová Dana , Donà Valentina , Euphrasie Daniel , Daffé Mamadou , Etienne Gilles , Manganelli Riccardo , Reyrat Jean-Marc
Published: September 2008

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that σ
F
is required for carotenoid pigment production in
Mycobacterium smegmatis
. We further show that a
sigF
mutant exhibits a transformation efficiency 10
4
-fold higher than that of the parental strain, suggesting that σ
F
regulates the production of components affecting cell wall permeability. In addition, a
sigF
mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the
M. smegmatis
genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.
Mechanism of 4-Nitrophenol Oxidation in
Rhodococcus
sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression
Authors: Takeo Masahiro , Murakami Masumi , Niihara Sanae , Yamamoto Kenta , Nishimura Munehiro , Kato Dai-ichiro , Negoro Seiji
Published: September 2008

4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil.
Rhodococcus
sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (
nphRA1A2
) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a
Rhodococcus
strain revealed that the transcription of
nphA1
is induced by only 4-NP (of several phenolic compounds tested) in the presence of
nphR
, which is constitutively expressed. Disruption of
nphR
abolished transcriptional activity, suggesting that
nphR
encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in
Escherichia coli
and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.
Passage through
Tetrahymena tropicalis
Triggers a Rapid Morphological Differentiation in
Legionella pneumophila
Authors: Faulkner Gary , Berk Sharon G. , Garduño Elizabeth , Ortiz-Jiménez Marco A. , Garduño Rafael A.
Published: September 2008

The intracellular bacterial pathogen
Legionella pneumophila
follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that
L. pneumophila
survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate
Tetrahymena tropicalis
before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through
T. tropicalis
, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene
rpoS
, or the response regulator gene
letA
, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through
T. tropicalis
. Therefore,
T. tropicalis
has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The
Tetrahymena
model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.
Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth inStreptomyces coelicolor
Authors: Hempel Antje Marie , Wang Sheng-bing , Letek Michal , Gil José A. , Flärdh Klas
Published: September 2008

Time-lapse imaging ofStreptomyceshyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape inStreptomyces.
MacAB Is Involved in the Secretion ofEscherichia coliHeat-Stable Enterotoxin II
Authors: Yamanaka Hiroyasu , Kobayashi Hidetomo , Takahashi Eizo , Okamoto Keinosuke
Published: September 2008

The heat-stable enterotoxin (ST) produced by enterotoxigenicEscherichia coliis an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments usingE. coliBL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, andyojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in themacABmutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in themacABmutant was restored by the introduction of pACYC184, containing themacABgene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.
Role of Two NtcA-Binding Sites in the Complex
ntcA
Gene Promoter of the Heterocyst-Forming Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Olmedo-Verd Elvira , Valladares Ana , Flores Enrique , Herrero Antonia , Muro-Pastor Alicia M.
Published: September 2008

Anabaena
sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N
2
in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the
ntcA
gene promoter from
Anabaena
sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the
ntcA
gene during heterocyst differentiation was studied in vivo by using an
ntcA-gfp
translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of
ntcA
in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.
YspM, a Newly Identified Ysa Type III Secreted Protein ofYersinia enterocolitica
Authors: Witowski Sarah E. , Walker Kimberly A. , Miller Virginia L.
Published: September 2008

Yersinia enterocoliticahas three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis ofY. enterocoliticaand is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersiniasecreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression ofyspMis regulated by temperature, NaCl concentration, and other known regulators of theysasystem. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain ofY. enterocoliticashows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed inSaccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspMY295) was expressed. Site-directed mutagenesis of the histidine of YspMY295ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).
Kinetic Analysis of Growth Rate, ATP, and Pigmentation Suggests an Energy-Spilling Function for the Pigment Prodigiosin ofSerratia marcescens
Authors: Haddix Pryce L. , Jones Sarah , Patel Pratik , Burnham Sarah , Knights Kaori , Powell Joan N. , LaForm Amber
Published: September 2008

Serratia marcescensis a gram-negative environmental bacterium and opportunistic pathogen.S. marcescensexpresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes forS. marcescensduring cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.
SciN Is an Outer Membrane Lipoprotein Required for Type VI Secretion in EnteroaggregativeEscherichia coli
Authors: Aschtgen Marie-Stéphanie , Bernard Christophe S. , De Bentzmann Sophie , Lloubès Roland , Cascales Eric
Published: September 2008

EnteroaggregativeEscherichia coli(EAEC) is a pathogen implicated in several infant diarrhea or diarrheal outbreaks in areas of endemicity. Although multiple genes involved in EAEC pathogenesis have been identified, the overall mechanism of virulence is not well understood. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS), has been identified in EAEC and most animal or plant gram-negative pathogens. T6SSs are multicomponent cell envelope machines responsible for the secretion of at least two putative substrates, Hcp and VgrG. In EAEC, two copies of T6S gene clusters, calledsci-1andsci-2, are present on thepheUpathogenicity island. In this study, we focused our work on thesci-1gene cluster. The Sci-1 apparatus is probably composed of all, or a subset of, the 21 gene products encoded on the cluster. Among these subunits, some are shared by all T6SSs identified to date, including a ClpV-type AAA+ATPase (SciG) and an IcmF (SciS) and an IcmH (SciP) homologue, as well as a putative lipoprotein (SciN). In this study, we demonstrate thatsciNis a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation. We further show that SciN is a lipoprotein, as shown by the inhibition of its processing by globomycin and in vivo labeling with [3H]palmitic acid. SciN is tethered to the outer membrane and exposed in the periplasm. Sequestration of SciN at the inner membrane by targeting the +2 residue responsible for lipoprotein localization (Gly2Asp) fails to complement ansciNmutant for SciD secretion and biofilm formation. Together, these results support a model in which SciN is an outer membrane lipoprotein exposed in the periplasm and essential for the Sci-1 apparatus function.
Characterization of a
Streptococcus
sp.-
Veillonella
sp. Community Micromanipulated from Dental Plaque
Authors: Chalmers Natalia I. , Palmer Robert J. , Cisar John O. , Kolenbrander Paul E.
Published: September 2008

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as
Streptococcus oralis
(RPS bearing) and
Streptococcus gordonii
(adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism
Veillonella
sp. strain PK1910, an oral isolate in our culture collection.
S. oralis
coaggregated with
S. gordonii
by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.
Mutations in Conserved Helix 69 of 23S rRNA of
Thermus thermophilus
That Affect Capreomycin Resistance but Not Posttranscriptional Modifications
Authors: Monshupanee Tanakarn , Gregory Steven T. , Douthwaite Stephen , Chungjatupornchai Wipa , Dahlberg Albert E.
Published: September 2008

Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of
Thermus thermophilus
selected for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, ΔmU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2′-O methylates C1920 of 23S rRNA. Inactivation of the
tlyA
gene in
T. thermophilus
does not affect its sensitivity to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations.
Characterization of Conserved Bases in 4.5S RNA ofEscherichia coliby Construction of New F′ Factors
Authors: Peterson James M. , Phillips Gregory J.
Published: September 2008

To more clearly understand the function of conserved bases of 4.5S RNA, the product of the essentialffsgene ofEscherichia coli, and to address conflicting results reported in other studies, we have developed a new genetic system to characterizeffsmutants. Multipleffsalleles were generated by altering positions that correspond to the region of the RNA molecule that interacts directly with Ffh in assembly of the signal recognition particle. To facilitate characterization of theffsmutations with minimal manipulation, recombineering was used to construct new F′ factors to easily move each allele into different genetic backgrounds for expression in single copy. In combination with plasmids that expressedffsin multiple copy numbers, the F′ factors provided an accurate assessment of the ability of the different 4.5S RNA mutants to function in vivo. Consistent with structural analysis of the signal recognition particle (SRP), highly conserved bases in 4.5S RNA are important for binding Ffh. Despite the high degree of conservation, however, only a single base (C62) was indispensable for RNA function under all conditions tested. To quantify the interaction between 4.5S RNA and Ffh, an assay was developed to measure the ability of mutant 4.5S RNA molecules to copurify with Ffh. Defects in Ffh binding correlated with loss of SRP-dependent protein localization. Real-time quantitative PCR was also used to measure the levels of wild-type and mutant 4.5S RNA expressed in vivo. These results clarify inconsistencies from prior studies and yielded a convenient method to study the function of multiple alleles.
Synergistic Contribution of theLegionella pneumophila lqsGenes to Pathogen-Host Interactions
Authors: Tiaden André , Spirig Thomas , Carranza Paula , Brüggemann Holger , Riedel Kathrin , Eberl Leo , Buchrieser Carmen , Hilbi Hubert
Published: September 2008

The causative agent of Legionnaires' disease,Legionella pneumophila, is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase.L. pneumophilaharbors thelqs(Legionella quorumsensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance inEscherichia coli. LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterizeL. pneumophilamutant strains lacking all four genes of thelqscluster or only thehdeDgene. While anhdeDmutant strain did not have overt physiological or virulence phenotypes, anlqsmutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis, and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of thelqsgenes into the chromosome of anlqsmutant strain. The deletion of thelqscluster caused more-severe phenotypes than deletion of onlylqsR, suggesting a synergistic effect of the otherlqsgenes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in thelqsmutant and wild-typeL. pneumophila. Genes involved in protein production, metabolism, and bioenergetics were upregulated in thelqsmutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome analysis revealed that a set of Icm/Dot substrates is not produced in the absence of thelqsgene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of thelqsmutant strain.
Alr0397 Is an Outer Membrane Transporter for the Siderophore Schizokinen inAnabaenasp. Strain PCC 7120
Authors: Nicolaisen Kerstin , Moslavac Suncana , Samborski Anastazia , Valdebenito Marianne , Hantke Klaus , Maldener Iris , Muro-Pastor Alicia M. , Flores Enrique , Schleiff Enrico
Published: September 2008

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacteriumAnabaenasp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.
Posttranslational Modification Influences the Effects of MgrA on
norA
Expression in
Staphylococcus aureus
Authors: Truong-Bolduc Que Chi , Ding Yanpeng , Hooper David C.
Published: September 2008

MgrA is a global regulator in
Staphylococcus aureus
. Differences in the effects of MgrA on
norA
expression have been reported for different strains, which varied in
rsbU
, a gene that affects the expression of
sigB
, which encodes an alternative σ factor involved in stress responses. We hypothesized that MgrA was modified by
sigB
-dependent factors that affected its ability to control the expression of the
norA
efflux pump. Heterologously expressed MgrA purified from
Escherichia coli
was incubated with crude extracts (CE) from strains RN6390 (
rsbU
) and SH1000 (
rsbU
+
) and tested for binding to the
norA
promoter. Purified MgrA exhibited greater binding to
norA
promoter DNA after being incubated with SH1000 CE than MgrA incubated with the RN6390 CE. Phosphorylation of MgrA occurring in cell extracts caused it to lose the ability to bind
norA
promoter DNA. Overexpression of
pknB
, encoding a candidate serine/threonine kinase, produced increased phospho-MgrA and led to a fivefold increase in the transcript level of
norA
for both RN6390 and SH1000, as well as a fourfold increase in the MICs of norfloxacin and ciprofloxacin for these two strains. The levels of expression of
pknB
in RN6390 and SH1000, however, indicated that additional factors related to
rsbU
or
sigB
contribute to the differential regulatory effects of MgrA on
norA
expression.
Modification of
Pseudomonas aeruginosa
Pa5196 Type IV Pilins at Multiple Sites with
d
-Ara
f
by a Novel GT-C Family Arabinosyltransferase, TfpW
Authors: Kus Julianne V. , Kelly John , Tessier Luc , Harvey Hanjeong , Cvitkovitch Dennis G. , Burrows Lori L.
Published: September 2008

Pseudomonas aeruginosa
Pa5196 produces type IV pilins modified with unusual α1,5-linked
d
-arabinofuranose (α1,5-
d
-Ara
f
) glycans, identical to those in the lipoarabinomannan and arabinogalactan cell wall polymers from
Mycobacterium
spp. In this work, we identify a second strain of
P. aeruginosa
, PA7, capable of expressing arabinosylated pilins and use a combination of site-directed mutagenesis, electrospray ionization mass spectrometry (MS), and electron transfer dissociation MS to identify the exact sites and extent of pilin modification in strain Pa5196. Unlike previously characterized type IV pilins that are glycosylated at a single position, those from strain Pa5196 were modified at multiple sites, with modifications of αβ-loop residues Thr64 and Thr66 being important for normal pilus assembly. Trisaccharides of α1,5-
d
-Ara
f
were the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues within the antiparallel beta sheet region of the pilin. TfpW was hypothesized to encode the pilin glycosyltransferase based on its genetic linkage to the pilin, weak similarity to membrane-bound GT-C family glycosyltransferases (which include the
Mycobacterium
arabinosyltransferases EmbA/B/C), and the presence of characteristic motifs. Loss of TfpW or mutation of key residues within the signature GT-C glycosyltransferase motif completely abrogated pilin glycosylation, confirming its involvement in this process. A Pa5196
pilA
mutant complemented with other
Pseudomonas
pilins containing potential sites of modification expressed nonglycosylated pilins, showing that TfpW's pilin substrate specificity is restricted. TfpW is the prototype of a new type IV pilin posttranslational modification system and the first reported gram-negative member of the GT-C glycosyltransferase family.
Regulation of the
Salmonella enterica std
Fimbrial Operon by DNA Adenine Methylation, SeqA, and HdfR
Authors: Jakomin Marcello , Chessa Daniela , Bäumler Andreas J. , Casadesús Josep
Published: September 2008

DNA adenine methylase (
dam
) mutants of
Salmonella enterica
serovar Typhimurium grown under laboratory conditions express the
std
fimbrial operon, which is tightly repressed in the wild type. Here, we show that uncontrolled production of Std fimbriae in
S. enterica
serovar Typhimurium
dam
mutants contributes to attenuation in mice, as indicated by the observation that an
stdA dam
strain is more competitive than a
dam
strain upon oral infection. Dam methylation appears to regulate
std
transcription, rather than
std
mRNA stability or turnover. A genetic screen for
std
regulators showed that the GATC-binding protein SeqA directly or indirectly represses
std
expression, while the poorly characterized
yifA
gene product serves as an
std
activator. YifA encodes a putative LysR-like protein and has been renamed HdfR, like its
Escherichia coli
homolog. Activation of
std
expression by HdfR is observed only in
dam
and
seqA
backgrounds. These data suggest that HdfR directly or indirectly activates
std
transcription. Since SeqA is unable to bind nonmethylated DNA, it is possible that
std
operon derepression in
dam
and
seqA
mutants may result from unconstrained HdfR-mediated activation of
std
transcription. Derepression of
std
in
dam
and
seqA
mutants of
S. enterica
occurs in only a fraction of the bacterial population, suggesting the occurrence of either bistable expression or phase variation.
Transcriptional Regulation of the Citrate Gene Cluster of
Enterococcus faecalis
Involves the GntR Family Transcriptional Activator CitO
Authors: Blancato Víctor S. , Repizo Guillermo D. , Suárez Cristian A. , Magni Christian
Published: September 2008

The genome of the gram-positive bacterium
Enterococcus faecalis
contains the genes that encode the citrate lyase complex. This complex splits citrate into oxaloacetate and acetate and is involved in all the known anaerobic bacterial citrate fermentation pathways. Although citrate fermentation in
E. faecalis
has been investigated before, the regulation and transcriptional pattern of the
cit
locus has still not been fully explored. To fill this gap, in this paper we demonstrate that the GntR transcriptional regulator CitO is a novel positive regulator involved in the expression of the
cit
operons. The transcriptional analysis of the
cit
clusters revealed two divergent operons:
citHO
, which codes for the transporter (
citH
) and the regulatory protein (
citO
), and upstream from it and in the opposite direction the
oadHDB-citCDEFX-oadA-citMG
operon, which includes the citrate lyase subunits (
citD
,
citE
, and
citF
), the soluble oxaloacetate decarboxylase (
citM
), and also the genes encoding a putative oxaloacetate decarboxylase complex (
oadB
,
oadA
,
oadD
and
oadH
). This analysis also showed that both operons are specifically activated by the addition of citrate to the medium. In order to study the functional role of CitO, a mutant strain with an interrupted
citO
gene was constructed, causing a total loss of the ability to degrade citrate. Reintroduction of a functional copy of
citO
to the
citO
-deficient strain restored the response to citrate and the Cit
+
phenotype. Furthermore, we present evidence that CitO binds to the
cis
-acting sequences O
1
and O
2
, located in the
cit
intergenic region, increasing its affinity for these binding sites when citrate is present and allowing the induction of both
cit
promoters.