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Transcriptional Regulator PerA Influences Biofilm-
Associated, Platelet Binding, and Metabolic Gene
Expression in Enterococcus faecalis
Scott M. Maddox1, Phillip S. Coburn2, Nathan Shankar2*, Tyrrell Conway1
1Advanced Center for Genome Technology, University of Oklahoma, Norman, Oklahoma, United States of America, 2Department of Pharmaceutical Sciences, The
University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections, traits facilitated by the
ability to quickly acquire and transfer virulence determinants. A 150 kb pathogenicity island (PAI) comprised of genes
contributing to virulence is found in many enterococcal isolates and is known to undergo horizontal transfer. We have
shown that the PAI-encoded transcriptional regulator PerA contributes to pathogenicity in the mouse peritonitis infection
model. In this study, we used whole-genome microarrays to determine the PerA regulon. The PerA regulon is extensive, as
transcriptional analysis showed 151 differentially regulated genes. Our findings reveal that PerA coordinately regulates
genes important for metabolism, amino acid degradation, and pathogenicity. Further transcriptional analysis revealed that
PerA is influenced by bicarbonate. Additionally, PerA influences the ability of E. faecalis to bind to human platelets. Our
results suggest that PerA is a global transcriptional regulator that coordinately regulates genes responsible for enterococcal
Citation: Maddox SM, Coburn PS, Shankar N, Conway T (2012) Transcriptional Regulator PerA Influences Biofilm-Associated, Platelet Binding, and Metabolic Gene
Expression in Enterococcus faecalis. PLoS ONE 7(4): e34398. doi:10.1371/journal.pone.0034398
Editor: Michael Chaussee, University of South Dakota, United States of America
Received November 28, 2011; Accepted February 27, 2012; Published April 4, 2012
Copyright: ? 2012 Maddox et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants AI48945 and GM095370 from the National Institutes of Health (nih.gov). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
As a commensal member of the intestinal microbiota, the
enterococci play an important role in establishing a healthy GI
tract and typically coexist in the host as a relatively small, yet
stable, population. Alternatively if the delicately balanced host/
commensal relationship is disrupted, if specific environmental cues
are detected, or if virulence traits are acquired, enterococci can act
as opportunist pathogens capable of multiple-site infections,
including infections of the heart, urinary tract, and bloodstream
[1,2,3]. In an effort to better understand the differences between
commensal and pathogenic enterococci, studies of pathogenic
enterococci increasingly seek to discover which traits promote
virulence, how these traits are inherited and what mechanisms are
used to coordinately regulate these traits to achieve pathogenicity.
While the enterococci have been known as infective agents for
more than 100 years [4], the majority of information regarding the
acquisition and deployment of virulence traits has been gathered
in the last few decades [5,6,7]. As a result of these studies, we have
a clearer picture of how the enterococci successfully transition
from a commensal to a pathogen. At the heart of this transition is
enterococcal promiscuity: the ease and frequency with which
many strains acquire and transmit mobile genetic elements
harboring loci that contribute to pathogenesis. In addition to
being intrinsically resistant to a broad range of antimicrobial
agents, enterococci have evolved resistance to many antibiotics by
acquiring plasmids or transposons comprised of genes that confer
resistance. Developing antibiotic resistance has increased the
pathogenic potential of the enterococci, as is evident by these
organisms becoming the leading cause of surgical site infections,
the second leading cause of bloodstream infections and the third
leading cause of nosocomial urinary tract infections [8]. Further-
more, antibiotic resistant strains are more likely to contain mobile
genetic elements that may harbor virulence traits [9]. Especially
problematic are strains that acquire both antibiotic resistance and
virulence traits, as the concurrence of these factors is correlated
with strains capable of producing infection outbreaks on a global
scale [10].
Facilitating the spread of virulence traits in a particularly
efficient manner are pathogenicity islands (PAI). PAI's are
characterized as clusters of genes encoding proteins with roles
involving transfer functions, virulence, stress survival, and
transcriptional regulation [11]. Furthermore these mobile genetic
elements can be distinguished from the native chromosome by a
significantly different G+C content [11]. While first discovered in
pathogenic Escherichia coli [12,13], these mobile genetic elements
are disseminated throughout many bacterial genera [11]. A
153 kb PAI consisting of 129 open reading frames was discovered
in Enterococcus faecalis MMH594 and shown to disperse to many E.
faecalis strains of various origins [10,14,15]. This PAI contains
many loci with roles in virulence, including esp (encodes
enterococcal surface protein), cytolysin toxin, and aggregation
substance, as well as factors potentially involved in horizontal
transfer and gastrointestinal tract colonization [14]. Esp is
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enriched among infection-derived isolates and has been shown to
increase in vitro biofilm formation [16,17]. The eight genes
comprising the cytolysin operon (cylR1, cylR2, cylLL, cylLS, cylMBAI)
form a two-peptide lytic toxin [18,19]. Cytolysin toxin is effective
against both prokaryotic and eukaryotic cells [20,21], and
contributes to mortality in various pathogenic models of infection
[22,23,24]. A pheromone-inducible aggregation substance (AS)
can also be found in many enterococcal strains. AS promotes
aggregation and conjugation [25,26], increases enterococcal
adherence to and uptake in eukaryotic cells [27,28] and increases
bacterial survival inside the macrophage [29].
Frequently, PAI's contain genes encoding transcriptional
regulators with various regulatory schemes, and the E. faecalis
PAI is no exception [11,14]. The E. faecalis PAI encodes an AraC-
type regulator, named PerA (for pathogenicity island-encoded
regulator) [14,30]. PerA is enriched among clinical E. faecalis
isolates and lies adjacent to the aforementioned PAI-encoded
virulence traits, which suggests PerA-dependent regulation of these
genes [14]. Through mutational analysis, we have previously
shown that PerA influences biofilm formation in a medium-specific
manner and contributes to virulence in a mouse peritonitis model
[30]. Additionally, the PerA-deficient strain was significantly
attenuated during macrophage survival, further supporting the
role of PerA as an important regulator of E. faecalis pathogenesis
Prompted by the observation that PerA coordinates E. faecalis
virulence in the mouse peritonitis infection model, we sought to
identify the genes that are regulated directly or indirectly by PerA.
We used Affymetrix GeneChip microarrays to experimentally
define the PerA regulon throughout exponential growth, upon
transition into stationary phase and during stationary phase
persistence. Our results suggest that PerA primarily regulates
genes located outside of the PAI in a growth phase-dependent
manner. These PerA-regulated genes are located throughout the
E. faecalis chromosome and include loci responsible for amino acid
metabolism, biofilm formation and phage-associated genes
putatively involved in platelet binding. Further experimentation
revealed that PerA influences the ability of E. faecalis to bind
human platelets and respond to the presence of bicarbonate.
Taken together with our previous findings [30], we interpret these
results to mean that PerA acts as a global transcriptional regulator
to coordinately regulate genes responsible for enterococcal
Overview of microarray data
PerA is an AraC-type transcriptional regulator that contributes
to pathogenesis in E. faecalis [30]. To define the PerA regulon,
transcriptional profiling was performed on E. faecalis E99 and an
isogenic DperA mutant strain (designated DBS01) using RNA
extracted from both strains at time points corresponding to mid-
600 nm,0.05, 0.5, and 1.0, respectively). The RNA was
reverse-transcribed and subsequently hybridized to E. faecalis
V583 genome microarrays. All array data shown are expressed as
ratios (DBS01 : E99) and considered to be significant if gene
expression was induced or repressed in the mutant strain greater
than twofold. The PerA regulon is extensive, as transcriptional
analysis revealed 151 genes differentially regulated.twofold
(log2=1) in DBS01 (Table S1). Of these 151 genes, 98 were up-
regulated and 53 were down-regulated. Nearly one-third (46 of
151) of the differentially regulated genes have unknown function,
20 are involved in metabolic functions, and 19 encode transport-
and stationaryphase(O.D.
related genes. Of the 98 up-regulated genes, 19 are up-regulated in
mid-exponential phase only, 6 are up-regulated in late-exponential
phase only, and 57 are up-regulated only in stationary phase
(Figs. 1A and 1B). Of the 53 down-regulated genes 10 are down-
regulated only in mid-exponential phase, 11 are down-regulated
only in late-exponential phase, and 27 are down-regulated only in
stationary phase (Fig. 1A). These data suggest that while PerA is
primarily a negative regulator, it can also act as a dual regulator, as
a positive influence on gene expression is also noted (Figs. 1A and
1B). Additionally, the PerA target genes show a high degree of
growth-phase dependent regulation, with the highest degree of
influence occurring in stationary phase (Figs. 1A and 1B). Finally,
we verified expression of perA in E99 using quantitative reverse
transcription PCR (qRT-PCR) and found that expression of perA
was maximal during stationary phase (data not shown); a finding
that reflects the high degree of PerA regulation at this time point.
DBS01 shows altered expression of PAI-related genes
The 153 kb PAI carries virulence determinants (including
cytolysin, Esp, and aggregation substance) adjacent to perA
[14,30]. The proximity of the perA gene to genes with ascribed
roles in virulence is suggestive of PerA regulation of PAI genes. In
DBS01, 5 PAI genes were differentially regulated in any of the
time points studied (Figs. 1B and 2). During mid-exponential
growth the EF0579 gene was induced (Fig. 1B). This locus encodes
a putative TetR-family protein with unknown function in E.
faecalis. Four genes encoding hypothetical proteins (EF0488,
EF0531, EF0532, EF0533) were down-regulated between 2 and
4 fold in DBS01 at late-exponential phase (Fig. 1B). In stationary
phase the EF0579 gene was again induced, while the EF0488 gene
was no longer differentially regulated (Fig. 1B). The microarrays
used in this study were developed using the strain V583 sequenced
genome. V583 is missing portions of the cytolysin operon, nsr and
gls24-like genes, and the entire esp gene due to a spontaneous 17 kb
deletion within the PAI [14]. Therefore, qRT-PCR was used to
determine the expression of these PAI genes found in strain E99
but absent in V583. qRT-PCR revealed no differential regulation
of these genes in DBS01 at any time point tested (data not shown).
The differential regulation of PAI hypothetical genes, but not
genes with previously ascribed roles in virulence, may indicate
PerA-dependent control of genes with an unknown function in
enterococcal pathogenicity; however this possibility remains to be
The transcription of many housekeeping genes is altered
in DBS01
AraC-type regulators are known to control a variety of cellular
processes, including metabolism and other housekeeping functions
[31]. We mined the transcriptome to determine if any house-
keeping genes were regulated by PerA, and found a number of
genes differentially expressed in DBS01. A number of genes
involved in basic cellular metabolism were down-regulated in
DBS01, including galK, rbsK (EF2961) and rbsD (EF2960) (Fig. 1B).
galK encodes for galactokinase, while rbsK and rbsD encode for
ribokinase and a ribose transporter, respectively, and are
potentially required for transport and metabolism of galactose
and ribose. Many housekeeping genes are induced in DBS01,
including genes encoding ribosomal proteins (rplQ, rpsP, rpsD
[EF3070], rpmB [EF3116] and rpmH [EF3333]) and pyrimidine
nucleotide biosynthetic genes (purA, EF0014) (Fig. 1B). Lastly,
putative peptide ATP-binding cassette (ABC) transporters were
significantly induced in DBS01. While poorly studied in E. faecalis,
these peptide transporters generally provide nutrients to bacteria
in the form of amino acids or short peptides [32,33]
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PerA regulates biofilm-related genes in E99
E. faecalis E99 is a urinary-tract isolate possessing a high biofilm
phenotype [34]. Recently a ubiquitous enterococcal locus was
characterized and named ebp [35]. The ebpABC operon encodes
the enterococcal biofilm-associated pilus and contributes to
endocarditis, urinary tract infections (UTI), and biofilm formation
[35,36]. The EbpABC proteins are polymerized through the
activity of Bps (formerly, SrtC), and together are required for
maximal biofilm production in E. faecalis [35]. EbpR acts as a
transcriptional activator of ebpABC and positively influences
biofilm formation [37]. As previously shown, the PerA regulator
influences E99 biofilm formation in a medium-dependent manner
[30]. To determine if PerA regulates ebpABC and bps gene
expression, we compared the transcriptome of DBS01 to E99
Figure 1. Comparisons of microarray results for E99 and DBS01. Control RNA was extracted from E99 and used to normalize the test RNA
extracted from DBS01 (DBS01 : E99). All data presented here are shown as fold change in gene expression (test : control). (A) Upper diagram: Venn
diagram comparing significantly up-regulated genes (.2 fold) in DBS01 during mid-exponential, late-exponential and stationary phase. Lower
diagram: Venn diagram comparing significantly down-regulated genes (.2 fold) in DBS01 during mid-exponential, late-exponential, and stationary
phase. (B) Hierarchically-clustered heat map of all genes differentially regulated.twofold between DBS01 and E99.
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during mid-exponential, late-exponential, and stationary phase. In
DBS01, the ebpABC operon and associated bps gene was induced
between 4 and 8-fold during mid-exponential and stationary
phases (average operon induction=6.2-fold) (Fig. 3). The
transition from mid-exponential to late-exponential growth was
concomitant with an increase in expression of the ebpABC operon
(average operon induction=8.8-fold) (Fig. 3). Induction of the
ebpABC and bps genes was confirmed by using qRT-PCR (Table 2).
The high degree of ebpABC up-regulation shown here, as well as
the increase in biofilm formation previously shown in DBS01 [30],
suggests that PerA may act as a repressor of the ebpABC operon
and associated bps gene in E99.
Next we sought to examine if PerA regulates other biofilm-
related genes found in E99, including esp, the bee locus, and
fsrABCD operon. esp encodes for enterococcal surface protein, a
high-molecular weight protein that has been shown to enhance
biofilm formation [16,38]. The bee locus is a unique five-gene
system that contributes to the high biofilm phenotype found in
E99 [34]. The microarrays used for this experiment were derived
from the E. faecalis V583 sequenced genome. V583 is missing the
esp gene due to a 17 kb PAI deletion [14], and does not contain the
conjugative plasmid harboring the bee locus (unpublished results).
Therefore it was impossible to examine gene expression of these by
using microarrays. qRT-PCR was used to determine possible
changes in gene expression for the esp and bee loci. When
comparing DBS01 and E99 using qRT-PCR, no significant
differential regulation of the esp or bee loci in any of the three
growth phases tested was observed (data not shown).
The fsr system, encoded by the fsrABCD operon, is similar to the
agrABCD operon found in Staphylococcus aureus [39]. Through the
production of gelatinase-biosynthesis activating pheromone, fsr
activates two genes encoding a gelatinase (gelE) and a serine
protease (sprE) resulting in biofilm formation [39,40,41,42,43].
Though little is known about the fsr or gelE-sprE loci in E99,
approximately 60% of E. faecalis clinical isolates produce gelatinase
[44]. We searched the microarray data and found no differentially
regulated genes in either the fsr or gelE-sprE loci in DBS01. Taken
together these data suggest that PerA may act to repress the
ebpABC operon and associated sortase while having little to no
influence on the expression of the esp, bee or fsr loci under the
conditions tested.
perA and ebpABC respond to the presence of bicarbonate
in E99
Using b-gal assays and qRT-PCR, Bourgogne et al. have
recently shown that E. faecalis OG1RF ebpABC expression increases
when grown in sodium bicarbonate in an ebpR-dependent manner
[45]. Our data suggest that PerA acts as a repressor of the ebpABC
locus (Figs. 1B and 3). Furthermore, AraC-type regulators are
known to respond to bicarbonate, including RegA in Citrobacter
rodentium and ToxT in Vibrio cholerae [46,47]. Given that OG1RF
lacks the E. faecalis PAI, including perA, we were curious to
determine the effects of bicarbonate on ebpABC expression in E99.
To do this we analyzed the transcriptome of E99 grown in THB
supplemented with 100 mM sodium bicarbonate. When com-
pared to E99 grown in THB, perA expression was down-regulated
in the presence of bicarbonate while ebpR (the activator of ebpABC)
was moderately induced (Fig. 4). Furthermore, the average ebpABC
expression increased approximately 7-fold (ebpA=8.0, ebpB=7.7,
ebpC=4.9), with the biofilm and pilus-associated sortase (bps) being
induced 4-fold (Fig. 4).
We reasoned that if PerA represses the ebpABC locus, a down-
regulation of perA in the presence of bicarbonate would cause a
response similar to that seen in DBS01 (DperA). When comparing
the transcriptome of E99 grown in THB supplemented with
100 mM sodium bicarbonate to DBS01 grown in THB, similar
trends in perA, ebpR-ebpABC and bps gene expression are observed
(Fig. 4). These results suggest that perA is down-regulated in the
presence of bicarbonate, concomitant with an induction of the
ebpR-ebpABC and bps loci.
Effect of the perA mutation on expression of ADI
The arginine deiminase (ADI) system is used by many
microorganisms to generate ATP via arginine fermentation [48].
Genes comprising the ADI pathway in E. faecalis are arranged as
the arcABCRD operon (ArcA, arginine deiminase, ArcB, ornithine
carbamolytransferase; ArcC, carbamate kinase; ArcR, Crp/Fnr
regulator, ArcD arginine/ornithine antiporter), and are known to
be transcribed in the presence of arginine [49]. The ADI operon
has a complex regulatory scheme with binding sites for two
arginine-sensitive regulators (ArgR1 and ArgR2), a catabolite
control protein (CcpA), as well as a protein involved in E. faecalis
pathogenicity (Ers) [49,50]. The regulatory roles of ArgR1 and
ArgR2 remain unclear, however multiple Arg boxes can be found
Figure 2. All genes differently regulated in DBS01 mapped
onto the E. faecalis chromosome. The outer ring displays those
genes differentially regulated during mid-exponential phase. The
middle ring displays those genes differentially regulated during late-
exponential phase. The inner ring displays those genes differentially
regulated during stationary phase. The innermost circle displays the
location relative to position zero in millions of base pairs of the E.
faecalis V583 genome. The locations of the arginine deiminase (ADI)
and enterococcal biofilm-associated pilus (Ebp) operons, the E. faecalis
pathogenicity island (PAI), and a phage related element are indicated.
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upstream of the ADI operon and data suggests that ArgR2 may
act as an arginine-specific signal transducer [49]. Furthermore,
expression of argR1 and argR2 increases in the presence of arginine
and is absent in glucose containing medium [49]. In DBS01 the
arcABCRD operon is highly up-regulated in all time points tested
(Fig. 3). On average, the arcABCRD operon is induced 7.6-fold
during mid-exponential growth and plateaus upon entrance into
late exponential phase induced 11-fold. The average expression of
the arcABCRD genes is up-regulated 3-fold during stationary phase.
This pattern of ADI pathway regulation is similar to that
previously observed in E. faecalis. Bourgogne et al. found that the
enterococcal FsrB transcriptional regulator negatively influences
arcABC expression during transition from exponential to stationary
phase; though it is unclear if this regulation is direct or indirect
[51]. Riboulet-Bisson et al. have shown that the Ers regulator
activates arcABC expression by binding upstream of the arcA gene
[50]. For unknown reasons and in contrast to this study, arcRD
gene expression was not differentially regulated by FsrB or Ers
[50,51]. In DBS01 argR1 gene expression was induced at all time
points tested (Fig. 3) while the argR2 gene was not differentially
regulated (data not shown). The argR1 and arcABCRD genes
account for 60% (6 out of 10) of the genes up-regulated in all time
points tested (Figs. 1A and 3), suggesting the PerA regulator may
act as a repressor of arginine catabolism in E. faecalis.
PerA regulates a putative temperate bacteriophage in
Temperate bacteriophages are disseminated throughout many
gram-positive bacteria, including E. faecalis. The E. faecalis V583
sequenced genome contains seven regions arising from integrated
phages [52]. Though the role of these phages in E. faecalis
virulence has yet to be discovered, each of these mobile elements
contains homologs of virulence determinants from Streptococcus mitis
phage SM1 [52,53]. We mined the microarray data for each of
these putative phage-related genes, and found a cluster of genes
similar to phage 04 in V583 that was differentially regulated in
DBS01 (Figs. 2 and 5). This element spans ef1985–ef2043 and
Figure 3. Plots comparing the log2expression ratios of the arginine deiminase (ADI) and enterococcal biofilm associated pilus
(Ebp) operons in DBS01.
Table 1. qRT-PCR primers used in this study.
PrimerSequence (59 - 39)
arcA-R AATGCCTGCAATCGCTTTTT
arcR-F TCCGAGAATCGGACTGTTTCA
ebpA-R CCAGGAACTGCTAATTCACGG
ebpB-F CGTACAGGCGGCAAGTCTTT
ebpB-R AGGTATTCCCCCGCTTGATT
bps-F CATTTCAGGCCATCGTGGTC
bps-R GCGTCTTCCCATTGACTTCG
Table 2. Members of the PerA regulon confirmed by qRT-
Gene Product0.05 0.5 1.0
ebpAvon Willebrand factor7.8 (0.07) 30.0 (0.05)21.1 (0.02)
ebpB Cell wall surface protein14.0 (0.03)30.0 (0.03) 19.7 (0.02)
ebpC Cell wall surface protein14.0 (0.1) 27.9 (0.06) 19.7 (0.1)
bpsSortase 2.6 (0.05)2.8 (0.06) 2.0 (0.01)
arcA Arginine deiminase30.0 (0.03)274.4 (0.1) 9.8 (0.03)
arcB Ornithine carbamoyltransferase24.3 (0.01)181.0 (0.3) 13.9 (0.04)
arcC Carbamate kinase8.0 (0.1) 73.5 (0.2)19.7 (0.05)
arcR Transcriptional regulator Crp/Fnr 4.3 (0.1)64.0 (0.1) 18.4 (0.08)
*Change in DBS01 gene expression (DBS01 : E99) at OD600=0.05, 0.5 and 1.0.
Experiments were repeated twice, with 3 replicates for each gene per assay.
Mean values shown (standard error in parenthesis).
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contains putative replication, integration and virulence functions.
The majority of genes on the phage display either no change or
non-significant induction or repression in DBS01 throughout all
growth phases. However a group of genes show significant growth
phase-independent repression in DBS01, including homologs of
pblA, pblB and a gene encoding a putative lysin (Fig. 5). PblA and
PblB mediate bacterial attachment to platelets in S. mitis [54]. The
lysin protein serves a dual purpose: permeablizing the bacterial cell
wall, thus permitting release of PblA and PblB, and binding to
platelets through interaction with fibrinogen and fibrinogen
receptors [55,56]. E. faecalis is known to aggregate human
platelets, yet the molecular mechanisms coordinating this process
have not been discovered [57]. The repression of pblA, pblB and
lysin in DBS01 suggests that PerA influences the expression of
genes putatively involved in platelet binding and cell wall
permeability residing on a temperature bacteriophage in E99.
PerA influences the binding to human platelets
PerA differentially regulates two distinct loci potentially
important in bacterial attachment to human platelets. First are
the putative pblA, pblB and lysin genes residing on a temperate
bacteriophage. Next is the Ebp pilus, which has recently been
shown to mediate bacterial attachment to human platelets [58].
Given that genes potentially involved in platelet binding were both
induced and repressed in DBS01 (the ebp and phage-related loci,
respectively), we sought to determine if DBS01 showed an altered
ability to bind human platelets. To assess this we compared the
ability of E99 and DBS01 to adhere to human platelets
immobilized in microtiter plates. As shown in Fig. 6, DBS01
binds human platelets significantly (P,0.0005, unpaired t-test)
better than the E99 wild-type strain. DBS01 bound platelets
approximately 5-fold better than E99. When DBS01 contained a
plasmid-encoded copy of perA (pGT101), platelet-binding abilities
were restored to the wild-type levels (Fig. 6). These results suggest
that the inactivation of perA increases platelet binding in DBS01,
possibly through the derepression of the ebpABC locus.
The perA gene is located on the E. faecalis PAI, adjacent to loci
with ascribed roles in virulence and genes with putative metabolic
functions [14]. Given its location, it was our hypothesis that the
primary function of PerA was to regulate the expression of PAI
genes in E. faecalis. However, transcriptional analysis revealed that
in DBS01 only 5 PAI genes of unknown function were altered in
gene expression during the time course study. To our surprise the
overwhelming majority of genes differentially regulated in the
DperA mutant were chromosomally located yet not residing within
the PAI. McBride et al. [10] have recently suggested that the
enterococcal PAI is comprised of clusters of genes that likely
undergo horizontal transfer as modules. Additionally, portions of
the enterococcal PAI have been shown to conjugatively transfer
both in vitro and in vivo [15]. These findings raise the possibility that
PerA is able to transfer to strains lacking the PAI and subsequently
exert alien control of native genes. In this scenario, the acquisition
of the transcriptional regulator PerA could effect a rapid
physiological change in the recipient. In Salmonella, HilD, a
transcriptional regulator encoded on the Salmonella pathogenicity
island SPI-1, has been shown to regulate genes on the evolutionary
distinct SPI-2 pathogenicity island [59]. Furthermore, E. coli strain
K12 genes can be regulated by Ler, a regulator located on the
locus for enterocyte effacement (LEE) pathogenicity island of
strain O157:H7 [60]. Our data suggest that PerA may have the
ability to control native chromosomal genes upon entry into a
recipient; however, the ability of PerA to transfer into an
enterococcal strain lacking the PAI and regulate native genes
remains to be tested.
Biofilm formation is often a key component of bacterial
pathogenesis [61,62,63]. Though not necessarily a virulence trait,
as biofilms are also produced by many avirulent bacteria, biofilms
contribute to pathogenicity by increasing resistance to antibiotics
and environmental stresses [64]. In E. faecalis, biofilms are
correlated with infective endocarditis [37] and urinary tract
infections [36], and promote bacterial survival inside phagocytes
Figure 4. perA, ebpR-ebpABC and bps gene expression in E99 grown in THB supplemented with 100 mM sodium bicarbonate (dark
bars) or DBS01 grown in THB (light bars). Microarray assays were performed twice. The values shown are mean expression intensities (mean 6
SD) of biological replicates. Fold changes in gene expression were calculated by comparing E99 grown in THB to E99 grown in THB+100 mM sodium
bicarbonate and by comparing DBS01 grown in THB to E99 grown in THB.
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[65]. PerA has been shown to influence biofilm formation in a
medium specific manner, as a perA-deficient strain designated
DBS01 produced more biofilm than the WT strain E99 [30].
Transcriptional profiling revealed that the enterococcal biofilm
associated pilus (ebp) locus, a ubiquitous determinant important for
biofilm production [35], was up-regulated in DBS01. This makes
possible the interesting scenario where the PAI-residing perA could
transfer to recipient strains and influence biofilm formation
through regulation of the ebp locus.
Expression of the ebp genes is controlled through multiple
transcriptional regulators. In addition to the PerA-dependent
repression of the ebp operon (Figs. 1B and 3), these genes are
activated through the action of EbpR [37]. More recently Gao et
al. have shown that expression of the ebp locus in E. faecalis
OG1RF is influenced by rnjB, a gene encoding RNase J2 [66].
OG1RF strains deficient in RNase J2 expression have reduced
ebpABC transcript levels and fail to produce Ebp pili, however the
regulatory mechanism responsible for these observations is
currently unknown [66]. Though poorly studied in E. faecalis,
Figure 6. Platelet binding activity of E99 and DBS01. The values shown are percent of wild-type (E99) binding (mean 6 SD). Differences in
platelet binding efficiencies were determined using an unpaired t-test (P=0.05). Platelet binding assays were performed in triplicate and each
experiment was repeated thrice (n=9).
Figure 5. Map of E. faecalis V583 phage 04. The putative proteins were compiled using the annotated V583 sequence. The direction of
transcription is shown in blue (reverse) and red (forward). Heat maps of expression ratios (fold change) for DBS01 are shown for mid-exponential (O.D.
600=0.05), late-exponential (O.D. 600=0.5) and stationary phase (O.D. 600=1.0).
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RNase J1 and J2 are highly conserved proteins encoded by rnjA
and rnjB, respectively [66,67]. In B. subtilis, the RNase J1 and J2
enzymes form heterotetramer complexes and are typically
involved in mRNA processing, stability and turnover [68,69].
We do not know if E99 possesses rnjA and rnjB, however these
genes appear to be ubiquitous as every E. faecalis sequenced
genome contain these loci [66]. Furthermore, we do not know how
PerA, EbpR, and RNase J1 and J2 are structured within the
regulatory network controlling Ebp pilus formation. It is possible
that RNase J1 and J2 function independently of either PerA or
EbpR. This would account for the absence of Ebp pili in EbpR
containing strains grown in pilus-inducing conditions [66].
E. faecalis is known to aggregate platelets [57] a phenotype
mediated, at least in part, by the Ebp pilus [58]. When comparing
the ability of DBS01 and E99 to bind human platelets, DBS01 was
found to adhere to platelets significantly (,5 fold) better than E99
(Fig. 6). This ability to bind platelets is frequently implicated in
promoting infective endocarditis [70,71]. When the heart valves
become damaged, platelet aggregation on the damaged tissue can
serve as binding foci for circulating bacteria. In animal studies,
these vegetations cause the further accumulation of platelets and
bacteria onto the infected surface, a condition that may lead to
heart failure or death [72].
PerA influenced the expression of a number of genes involved in
amino acid metabolism. The majority of these genes comprise the
ADI pathway (arcABCRD) in E. faecalis. The ADI pathway is used
by E. faecalis to produce ATP via arginine fermentation [73,74].
Expression of arcABCRD is tightly controlled as the ADI promoter
region contains multiple binding sites for transcriptional regulators
and catabolite repression elements [49]. Riboulet-Bisson et al. [50]
recently identified an Ers (enterococcal regulator of survival)
binding site upstream of the arcA gene, and suggested an activator
role for this protein. In the current work, microarray analysis
revealed that the ADI pathway is highly induced in DBS01, which
is suggestive of PerA repression of these genes. Of interest is the
increase in arcABCRD gene expression concomitant with the
induction of the ebp locus in DBS01 (Fig. 3). During an infection, it
is possible that these coordinately PerA-regulated genes perform a
related function. In the presence of host proteins or amino acids,
the de-repression of the arcABCRD operon would permit the
transport and degradation of liberated arginine. In this scenario
arginine fermentation may provide energy for biofilm formation
during pathogenesis. The biofilms could then serve to increase
bacterial persistence inside the host and further the invasion of
nutrient-rich host tissue. Furthermore, the PerA regulon comprises
genes encoding a putative peptide ABC transport system (Fig. 2).
These peptide transport systems provide nutrients to the cell by
internalizing amino acids and short peptides, and are often critical
for the survival of auxotrophic lactic acid bacteria [32]. Zhu et al.
[62] found that clinical isolates of Staphylococcus aureus selectively
extracted arginine from growth media during biofilm formation.
Chaussee et al. [75] found that in Streptococcus pyogenes the
expression of virulence factors is coordinately regulated with
amino acid catabolism. In this work, we show that PerA regulates
genes involved in amino acid catabolism and biofilm formation,
which further suggests a regulatory, if not functional, correlation
between amino acid degradation and biofilm formation. While
intriguing, the correlation between arginine metabolism and
biofilm formation in E. faecalis remains to be studied.
Bicarbonate production is important for maintaining pH
homeostasis in the small intestine, as it neutralizes acid in the
intestinal lumen and prevents damage to the adherent mucus layer
[76,77]. Many pathogens use the presence of bicarbonate as an
environmental signal to coordinate the expression of virulence
traits and frequently AraC-type regulators are involved [46,47,78].
Bourgogne et al. have shown that the transcription of the E. faecalis
OG1RF ebp locus is enhanced in the presence of bicarbonate, yet
the regulatory cascade linking bicarbonate to ebp expression is
unclear. In E99, PerA appears to be a repressor of ebpABC
expression (Figs. 1B and 3). In the presence of bicarbonate perA
was down-regulated concomitant with an induction of ebpR-
ebpABC and bps expression (Fig. 4). This suggests that in E99, PerA
may be part of the regulatory cascade controlling ebp expression in
response to bicarbonate whereby the production of bicarbonate in
the intestine causes a down-regulation of perA, which leads to the
production of the Ebp pilus. In this scenario, the sensing of
environmental bicarbonate ultimately stimulates the production of
an adhesin that could aid in colonization of the intestine.
From our data we are unable to determine if PerA directly
responds to bicarbonate or if it is influenced by other regulatory
mechanisms that either detect bicarbonate or are influenced by the
slight change in pH introduced by bicarbonate addition. AraC-
type regulators are comprised of a conserved C-terminal DNA-
binding domain and a N-terminal domain important for ligand
binding. Comparisons of the PerA sequence to other AraC-type
regulators that are known to detect bicarbonate (C. rodentium RegA
and V. cholerae ToxT) reveal that PerA exhibits C-terminus
similarity, yet virtually no N-terminus sequence similarity exists
(data not shown). Furthermore, we have previously shown that the
PerA N-terminus contains no similarities with other AraC-type
regulators [30]. It is possible that PerA senses bicarbonate using a
unique bicarbonate-binding motif, however it is also possible that
other regulators that sense bicarbonate may control perA
expression. In regards to the latter possibility, E. coli MarA and
SoxS are AraC-type regulators known to regulate transcription
without directly detecting a ligand [79,80].
PerA also appears to influence the expression of a number of
housekeeping genes. Perhaps most notably is the down-regulation
of genes in DBS01 involved in the basic metabolism of the cell,
concomitant with an induction of genes responsible for biofilm
formation and attachment to host cells (Fig. 1B). It is possible that
at the site of infection E99 uses PerA as a global dual-regulator to
orchestrate the down-regulation of many housekeeping genes non-
essential to pathogenicity while inducing genes responsible for
colonization and infection of the host.
We have previously shown that PerA contributes to E. faecalis
survival in the macrophage [30]. However, finding the PerA-
regulated genes that coordinate macrophage survival using our
current strategy has, thus far, proven inconclusive. We are keen to
realize the harsh phagosomal environment encountered by E.
faecalis during phagocytosis is almost certainly drastically different
than the conditions in this study. Though studies seeking to
determine the E. faecalis intracellular survival strategy have
increased our understanding of the challenges faced upon
phagocytosis, the whole-genome transcriptional response used by
E. faecalis during macrophage survival has yet to be revealed. This
information would not only yield a better understanding of the
phagosomal landscape during E. faecalis infection, but it would also
illuminate the E. faecalis macrophage survival strategy. During
intracellular survival, it is possible that basal (or perhaps enhanced)
expression of perA influences the transcription of hypothetical
function genes, thus impacting persistence within the macrophage.
In the current study we used whole-genome E. faecalis V583
microarrays to determine the PerA regulon in E99. Though we
used qRT-PCR to interrogate PAI genes in E99 that are missing
from V583, we realize there could be other genes present in E99
yet absent from the V583 microarray. E99 contains a large,
conjugative plasmid (pBEE99) comprised of genes that confer a
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Page 9
high biofilm phenotype and increased ultraviolet radiation
resistance [81]. Additionally pBEE99 contains genes putatively
encoding an aggregation substance and a two-component
bacteriocin [81]. Under the conditions tested PerA did not
regulate either the PAI genes or the bee locus. However, the
expression of the remaining pBEE99 genes in DBS01 remains to
be determined. Furthermore, since the E99 genome has yet to be
sequenced, this strain could possess unknown loci that are
potentially regulated by PerA and contribute to virulence.
In conclusion, our data suggests that PerA is a global
transcriptional regulator that coordinately controls genes impor-
tant for pathogenicity. We can now propose a mechanism of how
E99 achieves pathogenicity by using PerA as part of a regulatory
network controlling expression of virulence genes. When appro-
priate environmental signals are sensed (quite possibly the
presence of bicarbonate), the cell quickly and efficiently creates a
rapid physiological change by down-regulating one gene: perA. In
response to the environmental signal, the reduced levels of PerA
would alleviate repression of genes important for biofilm formation
and colonization of host tissues. Concurrently, metabolic and
substrate transport pathways critical for cell nutrition are induced
while unnecessary housekeeping genes are repressed, thus ensuring
the cell has the proper nutrients for pathogenicity.
Materials and Methods
Bacterial strains, media, and reagents
The strains used in this study were E. faecalis E99 [34] and an
isogenic DperA::ermR mutant (DBS01) [30]. The mutant DBS01
was complemented in trans as previously described [30]. The
strains were routinely cultured in Todd-Hewitt broth (THB)
containing 1% glucose or THB+1% glucose supplemented with
100 mM sodium bicarbonate when appropriate. Antibiotics used
for selection included kanamycin (25 mg/ml) and erythromycin
(50 mg/ml) (Sigma Chemical, St. Louis, MO). Growth was
monitored as absorbance at 600 nm using a Beckman-Coulter
DU800 spectrophotometer.
RNA isolation and Microarray analysis
RNA extraction and microarray analysis proceeded as previ-
ously described [82] with a few modifications. Briefly, strains E99
and DBS01 were grown at 37uC overnight in THB+1% glucose in
appropriate antibiotics. The bacteria were diluted 1:10,000 into
fresh, pre-warmed medium and incubated at 37uC. At predeter-
mined optical densities (600 nm; 0.05 for mid-exponential, 0.5 for
late-exponential, and 1.0 for stationary phase) cells were sampled
directly into ice-cold RNAlater (Ambion, Foster City, CA). Total
RNA was extracted using Qiagen RNeasy Minikits (Valencia, CA)
with optional on-column DNase treatment steps according to the
manufacturer's specifications. RNA integrity was checked by gel
electrophoresis and stored in 2 volumes of ethanol at 280uC.
cDNA was generated by first strand synthesis using Superscript II
(Invitrogen, Carlsbad, CA) and random hexamers according to the
manufacturer's specifications. Fragmentation and biotinylation of
cDNA proceeded according to the Affymetrix prokaryotic labeling
protocol using the ENZO Kit from Roche Diagnostics (Indianap-
olis, IN). Biotinylated cDNA was hybridized to custom Affymetrix
GeneChips for 16 h at 45uC. The custom microarrays used in this
study contained probes for several prokaryotic genomes including
Enterococcus faecalis V583 (GEO Accession number: GPL6702).
Affymetrix protocol ProkGE_WS2v2-450 was used to stain the
hybridized arrays. Following scanning, raw data files (.cel) were
analyzed using RMA processing with quartile normalization [83].
Biological and technical replicates were averaged, and genes were
considered to be significantly induced or repressed if the DBS01 :
E99 expression ratio was greater than twofold [84]. Heatmaps
were generated using DecisionSite for Functional Genomics
(Spotfire, Somerville, MA). The microarray data has been
deposited at GEO (GEO accession number, GSE31538). All data
are MIAME compliant.
Transcript levels were confirmed by qRT-PCR using RNA
extracted from cells harvested during mid-exponential, late-
exponential, and stationary phase. The primers listed in Table 1
were designed using Primer Express software provided with the
ABI Prism 7000 sequence detector (Applied Biosystems, Foster
City, CA). Amplicon lengths were 100 bp. Quantification of 16 S
rRNA levels was used as an internal control and to normalize
RNA. Amplification was detected using SYBR Green PCR Master
Mix (Applied Biosystems) with automatic calculation of threshold
value (CT). The fold changes in gene expression were determined
by comparing mRNA abundance in DBS01 to that in E99 as
previously described [85]. Analysis was repeated in triplicate on
two biological replicates for each time point. Replicates were
averaged and the results are presented in Table 2.
Assessment of Platelet Binding
The ability of E. faecalis cultures to bind human platelets was
assessed as previously described [55]. Briefly, human platelets were
washed, fixed and immobilized on poly-L-lysine-coated 22-mm-
diameter tissue culture wells at a concentration of 16108platelets
per well. Following 30 min incubation at 37uC, unbound platelets
were removed by aspiration. The remaining bound platelets were
subsequently incubated in a 1% casein solution for 1 h at 37uC to
reduce non-specific adherence. Following removal of the blocking
solution, each well was inoculated with 16108of E. faecalis E99,
DBS01, or DBS01 (pGT101) suspended in PBS and further
incubated with gentle rocking. After 1 h unbound bacteria were
removed by washing each well twice with PBS and the bound
bacteria were collected by scraping and resuspending them in
PBS. The number of bacteria bound to platelets was determined
by plating suspensions on THB supplemented with appropriate
antibiotics. Binding was expressed as a percentage of the
inoculum. Platelet binding assays were performed three times,
each assay replicated in triplicate (n=9) using blood from
multiple, healthy volunteers. Differences in platelet binding
efficiencies were determined using an unpaired t-test, as shown
in Fig. 6.
Ethics Statement
This study was performed under the supervision and approval
of the Institutional Review Board at the University of Oklahoma.
The platelets used in this study were purchased from Bioreclama-
tion (Long Island, NY) and obtained from a blood bank supplied
by healthy volunteers.
Supporting Information
Table S1
the E. faecalis E99 PerA regulon.
The gene, locus tag, and annotated gene product for
Author Contributions
Conceived and designed the experiments: SMM PSC NS TC. Performed
the experiments: SMM PSC. Analyzed the data: SMM PSC NS TC.
Contributed reagents/materials/analysis tools: SMM PSC NS TC. Wrote
the paper: SMM.
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PLoS ONE | www.plosone.org 11April 2012 | Volume 7 | Issue 4 | e34398


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A few years ago, Illinois' Medicaid program for the poor noticed some odd trends in its billings for group psychotherapy sessions.
Nursing home residents were being taken several times a week to off-site locations, and Medicaid was picking up the tab for both the services and the transportation.
And then there was this: The sessions were often being performed by obstetricians and gynecologists, oncologists and urologists — "people who didn't have any training really in psychiatry," Medicaid director Theresa Eagleson recalled.
So Medicaid began cracking down, and spending plummeted after new rules were implemented. In July 2012 the program stopped paying for group psychotherapy altogether for residents of nursing homes.
Yet Illinois doctors are still billing the federal Medicare program for large numbers of the same services, a ProPublica analysis of federal data shows.
Medicare paid Illinois providers for more than 290,000 group psychotherapy sessions in 2012 — more than twice as many sessions as were reimbursed to providers in New York, the state with the second-highest total.
Among the highest billers for group psychotherapy in Illinois were three OB-GYNs and a thoracic surgeon. The four combined for 37,864 sessions that year, more than the total for all providers in the state of California. They were reimbursed more than $730,000 by Medicare in 2012 just for psychotherapy sessions, according to an analysis of a separate Medicare data set released in April.
"That's not good," Eagleson said when told of the Medicare numbers.
Of the Illinois OB-GYNs billing for group psychotherapy, Dr. Josephine Kamper had the highest number of sessions. She was paid for 10,399 sessions in 2012, at a cost to Medicare of $207,980.
In 2011, the state Department of Financial and Professional Regulation placed Kamper on two years' probation for failing to evaluate a patient undergoing an abortion prior to anesthesia and failing to collaborate with a certified registered nurse anesthetist. The terms of her probation did not prohibit providing psychotherapy.
Efforts to reach Kamper for comment were unsuccessful.
Another OB-GYN, Lofton Kennedy Jr., billed for 9,154 group psychotherapy services. He declined to comment.
The third-highest-billing OB-GYN, Philip Okwuje, charged Medicare for 8,584 group therapy sessions. In a brief interview, he said he doesn't do them anymore. Okwuje was barred from Medicare and Medicaid from 2002 to 2005, records show, though the reason was not immediately available.
Thoracic surgeon Mark Lubienski said he began working with a company that offered group psychotherapy because he had to go on disability and could no longer perform surgeries. He had been experiencing episodes in which he temporarily lost consciousness.
Medicare paid $194,540 for more than 9,700 of his sessions in 2012, though Lubienski shared a tax form showing he received only about $52,000 from the company, Unified Therapeutics.
"I basically supervise social workers who run therapy sessions in intermediate and long-term care facilities for people who have psychiatric diagnoses," Lubienski said. "I'm there, I pitch in, I discuss things with the residents and stuff. We have a calendar of Medicare-approved topics that we go by."
And for residents who can't participate in the group sessions, "we see them individually."
Lubienski also was paid for 96 psychiatric interviews to diagnose mental illness. Lubienski said he doesn't see a problem with doctors who are not psychiatrists performing these services. "In certain states, it's mandated that the facilities have to offer these services yet there's nowhere near enough psychiatrists, psychologists, doctors, social workers to do it," he said.
Unified Therapeutics chief executive Todd Occomy said his company stopped providing the service in 2012 after the Medicaid changes in Illinois and has essentially ceased operations. "We were just getting squeezed," he said. "It got to the point where we could barely pay our physicians."
Aaron Albright, a spokesman for the U.S. Centers for Medicare & Medicaid Services, said in an email that Medicare has no policy regarding which physicians may perform group psychotherapy. During such sessions, "personal and group dynamics are discussed and explored in a therapeutic setting allowing emotional catharsis, instruction, insight, and support," according to rules set out by one of Medicare's contractors.

subset: commoncrawl


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White House Invites Retired NFL Coach Dungy to Join Advisory Council
Retired NFL coach Tony Dungy has been invited to join the White House's Advisory Council on Faith-Based and Neighborhood Partnerships, reported a spokesman for the former Indianapolis Colts head coach.
"He was invited and has not yet made a decision about whether or not he will accept," Todd Starowitz, a publicist at Dungy's book publisher, Tyndale House Publishers, told The Christian Post on Tuesday.
In total, there will be 25 members on the council, composed of religious and secular leaders and scholars from different backgrounds. Members of the council, each appointed to a one-year term, so far include prominent figures such as Dr. Frank S. Page, president emeritus of the Southern Baptist Convention; the Rev. Jim Wallis, president and executive director of Sojourners; Pastor Joel C. Hunter, senior pastor of Northland, a Church Distributed; and Richard Stearns, president of World Vision.
Only 15 of the council's 25 members have so far been confirmed to serve with the remaining 10 expected to be announced later this week, according to U.S. News & World Report.
The council will be responsible for advising the new White House Office for Faith-Based and Neighborhood Partnerships on how to direct government funds to religious and neighborhood groups engaged in social service work.
Regarding the invitation to Dungy, Peter Elliot, a veteran sports journalist who lived in Indianapolis for the past 10 years, said Obama made the right decision in tapping Dungy.
"His demeanor and adherence to living out his faith in all that he does makes him an instant cornerstone for the advisory council," wrote Elliot, a United Methodist, in his blog Wednesday.
The Rev. Barry Lynn, executive director of Americans United for Separation of Church and State, however, said news of Dungy's invitation was "unwelcome."
"This unnecessary body already has plenty of people on it who are way off the mark on civil rights and liberties. Dungy will be another," he commented on the multi-faith site Beliefnet.com.
"Dungy's fund-raising for the James Dobson-affiliated 'Indiana Family Institute' and his unequivocal opposition to same-sex marriage based on his view that God opposes it make him an unlikely candidate for useful discussion of faith based partnerships and civil rights, one of the key issues the panel will hammer out," Lynn added.
When the revamped White House office was unveiled in February, President Obama said it would work with nonprofit organizations "both secular and faith-based" and would help them determine how to make a bigger impact in their cities, learn their obligations under the law and cut through government red tape.
Obama said the top priority of the office will be "making community groups an integral part of our economic recovery and poverty a burden fewer have to bear when recovery is complete."
Dungy, the first African American head coach to win the Super Bowl, is widely known for his outspoken Christian faith, which has influenced his work on and off the field. Aside from winning six division titles and appearing in three conference championship games, Dungy was involved with a number of charitable organizations and urged players and coaches to get involved in community work, too.
After announcing his retirement earlier this year, the championship winning coach said he was not sure exactly what new goals he'd be pursuing but was looking forward to devoting his extra time to his family and the social causes he embraces.
"I think I've got a responsibility to be home a little bit more, be available to my family a little bit more and do some things to help make our country better," Dungy said. "I don't know what that is right now, but we'll see."
Some of the charitable organizations that Dungy has been involved with in recent years include the Fellowship of Christian Athletes, Athletes in Action, Mentors for Life, Big Brothers Big Sisters, Boys & Girls Clubs, the Prison Crusade Ministry, and All Pro Dad.
He has also worked with Basket of Hope, the Black Coaches Association National Convention, Indiana Black Expo, the United Way of Central Indiana, and the American Diabetes Association.


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Confused: PL-259 for VHF?
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You might want to take a look at mini-UHF connectors. They come in male and female, are low profile and easy to install with the right crimper. The female is the third one down in this photo I took of different connectors I installed in my shop.
There is nothing wrong with crimp connectors, they work just as well (except for the center-pin type which don't work well), as soldered if done properly. I'm a pro and use both types all the time. Mouser sells a pretty good
crimp kit
for a very good price $79. A little high for the casual user maybe but still a great price for what you get. I have all the expensive amphenol crimpers/dies $$$$ as well as this kit from Mouser. It's a good quality crimp/die kit.
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Target is expected to see a 3.7% gain in same-store sales, in line with the 3.6% increase expected across all retail stores for the month, according to the Thomson Reuters Same-Store Sales Index. Early holiday sales and a strong Cyber Monday are credited with the positive news.
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Memorial To Fix Martin Luther King, Jr. Quote
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Good morning. I'm Renee Montagne. Americans honor Martin Luther King, Jr. today. And those visiting his new 30-foot tall memorial in Washington, D.C. will find a quote that reads: I was a drum major for justice, peace and righteousness. Well, it may be carved in granite, but it's not actually what the civil rights leader said. Fixing a quote embedded in three feet of stone presents quite a challenge, but it will be changed to more accurately honor Dr. King's memory. It's MORNING EDITION. Transcript provided by NPR, Copyright NPR.


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Latest Jiangsu Province Stories
Biaoxin Mixers included the BXZRH vacuum homogenizing mixer in their popular line of mixers.
Biaoxin Mixers employs a team of highly qualified engineers whose primary job is to manufacture the highest quality machinery used for industry mixing.
Biaoxin is proud to display the latest vacuum multi-shaft mixers which use a vacuum processing system to produce paste and suspensions which are used in the food industry. Xuyi,
Biaoxin Machinery is one of the world's leading companies which manufacture lab mixers in their facility.
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Biaoxin Mixers was recently contacted by a cosmetics company from Tanga, Tanzania, East Africa. The company ordered and inspected the 7T petroleum jelly mixers.
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Call of Duty Modern Warfare 3 FAQs released
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Re: Call of Duty Modern Warfare 3 FAQs released
I see where your coming from, i am cod veteran myself but i have to say of all the PC CoD's i would put MW2 as the worst...It had potential but the lack of support for the PC version was the final straw. I had high hopes for MW3 because of the promises made but tbh it is sugar coated...
If you enjoyed past MW SP modes there is no doubt in my mind you will enjoy MW3, however speaking primarily of 'public' MP i dont see enough of a change from MW2...it seems that everything that made MW2 cack for PC has returned in MW3. IMO CoD4 PC will never be beaten by any other cod in terms of MP gameplay both public and competitive...
I will be keeping an eye out on how MW3 pans out for competitive play but with the locked cvars that allowed us to make cod4 so silky smooth with almost perfect bullet registration i can't see it taking off like CoD1, CoD2 or CoD4.
Edit: Actualy imo W@W was the worst cod for me, even with the consolitis of MW2 the gameplay was better than W@W.
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text: What's in store for Formula 1 as the second turbo era looms? 13 January, 2014 The turbo on Michele Alboreto's Ferrari 156/85 blows up in the pits during the 1985 British GP at Brands Hatch – turbo failures at the time were dramatic First, a question: who was the last F1 driver to win a World Championship Grand Prix in a turbo-charged car? (answer at end). Second, a history lesson: turbo-charged engines went out at the end of the 1988 season, when McLaren used Honda power to make Ayrton Senna the World Champion for the the great Brazilian's time and to claim the Constructors' Championship on what was the fourth occasion in the team's history to that date. Obviously that means that none of the current drivers have any experience of racing in F1 with turbo-charged engines, which are the new norm for 2014. The Energy Recovery Systems, a huge step up from the KERS we had come to know, will deploy twice the power and have 10 times the capacity of the previous technology, allowing the driver to call on an extra 120 kilowatts of power for more than half a minute on every racing lap. Renault F1 turbo engine for 2014 Through the 64-year history of the World Championship a total of 12 engine suppliers have powered the title-winning drivers. But in 2014 only three companies will be supplying engines to the sport's 11 teams: Ferrari, Mercedes and Renault. Of those, only Ferrari and Renault have previous F1 experience with turbo-charging – Mercedes retired from the sport way back in 1955 and returned only in 2010, well into the V8 normally aspirated era which began in 2006. Third, today's vocabulary practice: back in the late Eighties all the talk was of turbos, boost, wastegates and throttle lag, each of which was responsible at various times for team and driver frustrations as they all got to grips with the technology of the time. Turbo/engine failure was perhaps the most common reason given for retirements. Jacques Laffite suffers a turbo engine failure during the 1986 British GP As the great Alain Prost has said, "You have to say that the turbo engine years generated interest in F1: everyone was interested in this new technical challenge. It was also a bit of an emotional journey, insofar as huge developments were expected at each race." Will we see the same hair-tearing at the start of 2014 when the radical new F1 comes to Melbourne? Probably not – the issues raised by the technology a quarter of a century ago have been largely by-passed. On the other hand, there is a new generation of complex technology to be mastered: two turbos at work to provide the 'boost' in engine power which is the technology's whole raison d'être. The Honda RA168-E V6 turbo engine fitted to the 1988 McLaren MP4/4. Boost – pressurizing the engine to more than its nominal cylindrical capacity – also equates to fuel use, and fuel flow is one of the central elements of the new power-train technology in F1. It's limited to 100 kilogrammes of racing fuel per hour, and the total fuel allowed is 100 kilos per grand prix. The fuel flow limit puts a check on the amount of boost that can be used. We don't have wastegates any more, either: they were escape exits for excess energy back in the day. Nowadays the turbo will have a second electric motor attached to it to convert that excess energy into instantly usable electricity. That in turn means that throttle lag shouldn't be a problem, as it used to be, with drivers desperately waiting for what old-timers like Derek Warwick used to call that 'kick in the back' that propelled the cars forward at breakneck speeds. Mercedes V6 turbo F1 engine But how well will the three engine-suppliers succeed in recuperating energy, storing it and deploying it effectively? Many expert analysts believe that, following the bullet-proof reliability of engines we have come to expect in recent years, engines will not only make the difference but may also be the weakest link in the chain of components that contribute to a grand prix car's performance. "It's a return to an era when the driver will need to be strategic and very calculating in how he uses his racing car," adds Prost. "Being quick will no longer be enough on its own: you'll need to be quick and sensitive." And that should suit a certain young German down to the ground… Ferrari have now contested a staggering 870 grands prix, winning an equally amazing 222 of them. But there were only two wins for the Scuderia in 2013 – both for Fernando Alonso, and there has been some fairly loud sabre-rattling from Di Montezemolo ahead of the new F1 era. The Italian marque will supply not only the scarlet cars of the Scuderia, but also those of Sauber and Marussia. Gilles Villeneuve turbo powreed Ferrari 126C2 during the 1982 South African GP Mercedes will cater for four teams: the works cars, of course, plus McLaren, Force India and Williams. Teams like Sir Frank's will be desperately hoping that wholesale changes level the playing-field and allow the independents like Williams back into the race, so to say. Renault, meanwhile, brings a brilliant record of success in the last two decades – 12 Constructors' titles and 11 Drivers' titles between 1992 and 2013 – to their pursuit of continued glory in the new era. Can four straight titles with Red Bull become five with the new power trains? Alain Prost drives to victory at the 1988 Australian GP – the final F1 victory for a turbo powered car at the end of the first turbo era With so much change in the offing, any degree of continuity can only be a positive. That's why Mercedes looks such an attractive bet: the only team with the same drivers as it had in 2013, both Rosberg and Hamilton operating at a high level, and a genuine ambition to build on last year's runners-up status. But if you can't make up your mind, hang fire for another year. In 2015 Honda will return yet again to F1, where they bridged the end of the turbo era and the return to normally aspirated engines with five straight title wins from 1987 through 1991. You can bet that they're coming back intending to win… The sport's last turbo charged winner was Alain Prost, winner of the 1988 Australian Grand Prix in Adelaide driving a McLaren Honda. (Report by Australian GP Media Office) Subbed by AJN. Content on GrandPrix247.com by: staff & contributors, Reuters syndication, GMM service, Getty Images, Formula 1 teams, sponsors & organisations.


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All day Thursday and Friday, a diverse group of engineering and IT experts are gathering together to explore emerging research needs for cybersecurity in cyber-physical systems. The two-day workshop is sponsored by the Cyber Security Research Alliance and National Institute of Standards and Technology. Donna Dodson is division chief of the Computer Security Division and the acting chief cybersecurity Advisor at NIST.
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If you suspect your employees are grumbling behind your back, you might be right. A new analysis shows more feds think their leaders have room for improvement. Deloitte and the Partnership for Public Service have issued a report based on the most recent Federal Employee Viewpoint Survey.
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Remember when defense contractors were threatening to layoff employees because sequestration would be so tough on them? Turns out the budget cuts haven't been too bad, so far. Defense contracting actually shot up last month. What's going on? For some answers we turn to Bloomberg Government defense reporter Nick Taborek.
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Defense Secretary Chuck Hagel says sequestration is harming military readiness. But in his first policy address since taking office, Hagel said the Defense Department is preparing for a future with fewer resources. As Federal News Radio's Jared Serbu reports, Hagel is planning major reforms to make the military less expensive to operate.
Two central themes emerged from the first listening session to implement President Obama's executive order on cybersecurity. The first was obvious: collaboration must underlie the entire process to create the cybersecurity framework. But it was the second theme that will set the path forward for how the cyber framework will be developed by and for critical infrastructure providers. Federal News Radio's Jason Miller reports on with details on the emerging roadmap to implement the cyber executive order.
Cybersecurity Update
- The Homeland Security Department has put the cybersecurity executive order into high gear. DHS has created eight working groups to begin work on implementing the two-month old plan to improve the information assurance of critical infrastructure providers. DHS deputy secretary Jane Lute says the groups bring together public and private sector experts to work on topics such as incentives for adoption of the cyber framework, collaboration, planning and evaluation, and privacy and civil liberties assessments. Lute calls this effort an ambitious program on an ambitious timeline. The executive order set an October deadline for the first draft of the cyber framework to be completed. (Federal News Radio)
- Cyber workforce avoiding retirement wave -- for now (Federal News Radio)


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By Seth Green: Light middleweight contender Jamie Moore (31-3, 22 KOs) defeated Michelle Piccirillo (50-5-1, 29 KOs) in a 3rd round stoppage in a scheduled 12-round bout for the vacant EBU light middleweight title tonight at the Robin Park Centre, Wigan, Lancashire.
Moore,30, dropped Piccirillo three times in the fight - two times in the 2nd and one more time in the 3rd with a left to the body, sending Piccirillo to the canvas for the final time. Piccirillo spit out his mouthpiece and grabbed the top ropes with both hands while on the canvas, trying to get oxygen and recover from the big left hand. The fight was then stopped by referee Robin Dolpierre at 2:15 of the round.
Piccirillo, 39, was never in the fight due to his lack of power which prevented him from being able to mix it up with Moore. Without any power shots to fall back, Piccirillo futilely attempted to keep Moore off of him with jabs alone. It didn't work. Moore ducked under most of Piccirillo's jabs and once inside he hammered him with hard left hooks and rights to the head. In the 1st round, Moore hurt Piccirillo with a hard left to the body in the last seconds of the round causing Piccirillo to retreat backwards in pain.
In the 2nd round, Moore landed a cuffing right hand to the back of Piccirillo's head causing him to go down. At the same time that he threw the punch, Moore's feet got tangled with Piccirillo's, both the cuffing shot and the tangle of feet caused the knockdown. The referee, however, totally missed it and mistakenly ruled it a knockdown when it shouldn't have been. Late in the 2nd round, Moore hurt Piccirillo with a left hook to the head, and immediately poured in another left and then a right to drop Piccirillo to the canvas.
In the 3rd, Piccirillo seemed to get his feet under him and fight much better, using his jab to nail Moore on the outside. However, a minute into the round Moore staggered Piccirillo with a left hook to the jaw. A short while later, Moore nailed Piccirillo with a left to the body that caused Piccirillo to turn around and walk a couple of steps and go down on his knees in the corner, spitting out his mouthpiece at the same time. The fight was then stopped by the referee.
In other action, former 2008 Irish Olympian Darren Sutherland (2-0, 2 KOs) obliterated his overmatched opponent Siarhei Navarka in the 3rd round of a scheduled six round super middleweight bout with a series of power shots. Sutherland, 26, battered Nevarka, 29, throughout, hammering him with hard left hooks and right hands to the head. In the 3rd round, with Navarka taking nonstop punishment, referee Phil Edwards stepped in and halted the fight at 0:26 of the round.
Sutherland threw mostly single shots in the first two rounds of the fight, but in the 3rd, Sutherland began unloading on Navarka with powerful combinations, hurting him. The referee then stepped in and halted the slaughter. Sutherland looked extraordinary throughout the fight, landing hard shots that would have caused most super middleweights a lot of problems.
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Edited by L&B's Mama, 20 March 2012 - 09:03 AM.
Question about tank cage.
Started by L&B's Mama , Mar 20 2012 09:02 AM
6 replies to this topic
Posted 20 March 2012 - 09:02 AM
So I just moved Lucy and her miniature clan into the new 20 or 29 gallon tank and the babies love all the new roaming space. I put new bedding, along with some bedding from the old cage. My question is she's been digging in the corners, which I know is because she's trying to find a good place for a new nest, but she keeps biting on the enamel coating stuff that is used to seal the seams of the glass together in one of the corners. Is this going to harm her in any way? If not, will she just stop chewing on it on her own, or is there something I can do to help stop it?
Posted 20 March 2012 - 03:29 PM
First, I'm really glad they are loving all the new space! On to the question... well, as you know, you can't stop a hamster what it wants to do, they have a mind of their own. So, what I would do is try making a nest for her, or put her nest in a box or a hideout. Or, if that doesn't work try blocking it of with maybe some cardboard. I hope this helps!
Posted 20 March 2012 - 08:28 PM
I did make a nest for her because she was so busy chewing it that the babies tried to make a nest for themselves in the food bowl lol. My next investment for her is a little hideout. I shall try the cardboard idea. Thank you
Posted 21 March 2012 - 12:58 AM
Nope, that glue is nontoxic so it won't harm her. Silly hams! My old hamster when I had a tank awhile back used to do the same thing. Haha, they're like obsessed with that glue stuff. A hideout should help, but some hammies don't use them. It all depends. Try giving her a plain Milkbone to chew, it may prevent her from chewing the glue stuff.
Posted 21 March 2012 - 01:24 AM
Well I threw three chew sticks in there and it seems to help. She seems to be fond of the blue chew stick versus the others lol.
Posted 21 March 2012 - 02:24 AM
You might to try a simple Kleenex box rather than buying a hideout. My hamsters all shun the hideouts when I give them a Kleenex box. It's quite funny.
Posted 21 March 2012 - 02:27 AM
I believe I have a Kleenex box. Thanks I'll try that


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1. Cut the stalks of the asparagus in thirds.
2. Place the tips in a steamer and steam until tender. Set them aside for garnishing your soup.
3. In a pot melt the butter and add the onion. Cook for 6 minutes until softened and translucent.
4. Put the cut asparagus and sauté for 4 minutes.
5. Pour the stock. Simmer for about 15 minutes until the asparagus is cooked through and tender.
6. Using an immersion blender, blend the soup until creamy.
7. Return the soup to the pot and add the nutmeg and cream. Season with salt and pepper to taste.
8. Heat soup through turn off the heat.
9. In a serving bowl, serve this soup garnished with asparagus tips.
If you do not have an immersion blender, wait for the soup to cool down and blend it. Hot liquids can explode through the blender lid so be careful when dealing with them.


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However, it will not be able to accommodate all the traders from George Town
The design of the new wholesale foodgrain market at Koyambedu will be similar to that of the existing fruit and vegetable market. The new market structure will have ground plus one floor with chiselled dressed granite cladding, a sloped roof and decorative tiles.
The Chennai Metropolitan Development Authority on Tuesday cleared the finalisation of tender for construction of the market at a cost of Rs.69.17 crore with similar design. The contractor will begin the construction work by January 1, said an official of CMDA.
The market will start functioning by June 2014.
However, as the market is coming up at an alternative site between B-Road and E-Road in Koyambedu with the land originally earmarked for the project being taken over by Chennai Metro Rail, all the traders who wish to relocate will not get shops as the demand is four times that of proposed shops in the existing design of the market.
Even though the administrative sanction for the market was given in May, the tendering process took time on account of various issues pertaining to the demand of traders in George Town, who will have to shift businesses from the existing wholesale foodgrain market in George Town to Koyambedu.
As the new structure is located close to the upcoming metro station in Koyambedu, residents and small businesses will be able to purchase products such as cereals, pulses and spices at an affordable cost. However, the project covering 364 shops measuring 280 sq ft, 92 shops measuring 740 sq ft and 36 shops measuring 1,200 sq ft will not be enough to meet the demand.
The land available with the CMDA is limited because of the metrorail land acquisition and there is no scope for expansion of the market in future in Koyambedu, said officials.
The project is aimed at decongesting the central business district of George Town by shifting the business to Koyambedu where phase-I of the market for vegetables, flowers and fruits has over 3,194 shops
According to CMDA officials, Over 2,000 of the traders are part of the bustling market of foodgrains and spices in Kothwal Chavadi.
The total area of the built up space of the market will be 2,13,200 sq ft. Some traders have already paid a portion of the shop cost and those who get the shops will pay the rest in instalments, said an official.