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Research ArticleMicrobiology
Gene expression kinetics governs stimulus-specific decoration of the Salmonella outer membrane
Xinyu Hong1,2,3,4,
H. Deborah Chen2,*, and
Eduardo A. Groisman2,3,†
1Department of Cell Biology, Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
2Department of Microbial Pathogenesis, Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
3Yale Microbial Sciences Institute, P.O. Box 27389, West Haven, CT 06516, USA.
4Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06536, USA.
↵†Corresponding author. Email: eduardo.groisman{at}yale.edu
↵* Present address: Translational Medicine Research Centre, Merck Sharp and Dohme, 8 Biomedical Grove, #04-01, Singapore 138665, Singapore.
Science Signaling 08 May 2018:
Vol. 11, Issue 529, eaar7921
DOI: 10.1126/scisignal.aar7921
Xinyu Hong
Department of Cell Biology, Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.Department of Microbial Pathogenesis, Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.Yale Microbial Sciences Institute, P.O. Box 27389, West Haven, CT 06516, USA.Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06536, USA.
ORCID record for Xinyu Hong
H. Deborah Chen
Department of Microbial Pathogenesis, Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
Eduardo A. Groisman
Department of Microbial Pathogenesis, Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.Yale Microbial Sciences Institute, P.O. Box 27389, West Haven, CT 06516, USA.
ORCID record for Eduardo A. Groisman
For correspondence: eduardo.groisman@yale.edu
Same genes, different phenotypes
The lipopolysaccharide (LPS) coat of Gram-negative bacteria can be modified to evade the host immune response or confer resistance to antimicrobial compounds. Modifications that reduce the negative charge of the LPS component lipid A increase the resistance of Salmonella to the antibiotic polymyxin B and other cationic antimicrobial compounds. Hong et al. found that different stimuli elicited distinct lipid A modification profiles in Salmonella enterica serovar Typhimurium, although they promoted expression of the same set of genes encoding lipid A–modifying enzymes. Different stimuli caused the genes to be activated with different kinetics, thus altering the temporal order in which the lipid A–modifying enzymes were produced. This resulted in distinct lipid A profiles and different effects on resistance to polymyxin B. These findings illustrate how bacteria can use a limited set of enzymes to generate a range of adaptations to different stimuli.
Lipid A is the innermost component of the lipopolysaccharide (LPS) molecules that occupy the outer leaflet of the outer membrane in Gram-negative bacteria. Lipid A is recognized by the host immune system and targeted by cationic antimicrobial compounds. In Salmonella enterica serovar Typhimurium, the phosphates of lipid A are chemically modified by enzymes encoded by targets of the transcriptional regulator PmrA. These modifications increase resistance to the cationic peptide antibiotic polymyxin B by reducing the negative charge of the LPS. We report the mechanism by which Salmonella produces different lipid A profiles when PmrA is activated by low Mg2+ versus a mildly acidic pH. Low Mg2+ favored modification of the lipid A phosphates with 4-amino-4-deoxy-l-aminoarabinose (l-Ara4N) by activating the regulatory protein PhoP, which initially increased the LPS negative charge by promoting transcription of lpxT, encoding an enzyme that adds an additional phosphate group to lipid A. Later, PhoP activated PmrA posttranslationally, resulting in expression of PmrA-activated genes, including those encoding the LpxT inhibitor PmrR and enzymes responsible for the incorporation of l-Ara4N. By contrast, a mildly acidic pH favored modification of the lipid A phosphates with a mixture of l-Ara4N and phosphoethanolamine (pEtN) by simultaneously inducing the PhoP-activated lpxT and PmrA-activated pmrR genes. Although l-Ara4N reduces the LPS negative charge more than does pEtN, modification of lipid A phosphates solely with l-Ara4N required a prior transient increase in lipid A negative charge. Our findings demonstrate how bacteria tailor their cell surface to different stresses, such as those faced inside phagocytes.
Gram-negative bacteria are surrounded by an outer membrane that confers protection from various toxic agents (1). The outer membrane is composed of phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The innermost portion of the LPS is lipid A, followed by a central core region and the outermost O-antigen (2, 3). Phosphate residues in the lipid A and inner core are responsible for the negative charge of the LPS at neutral pH. This negative charge is normally neutralized by divalent cations, primarily Mg2+. However, certain bacteria chemically modify their LPS in response to specific environmental signals (2, 4), thereby altering permeability (5, 6), resistance to antibacterial compounds (7–10), and activation of the immune system of host animals (2, 11).
The Gram-negative bacterium Salmonella enterica serovar Typhimurium chemically modifies its lipid A phosphates with 4-amino-4-deoxy-l-aminoarabinose (l-Ara4N) and phosphoethanolamine (pEtN) (Fig. 1A) when experiencing conditions that activate PmrA, a transcriptional activator of genes encoding lipid A–modifying enzymes and an inhibitor of one such enzyme (4, 12). PmrA decreases the negative charge of lipid A in several ways. First, it promotes transcription of the pbgP operon and the ugd gene, which are responsible for the modification of the lipid A phosphates with l-Ara4N, and of the pmrC gene, which mediates modification of the lipid A phosphates with pEtN (9, 13) (pbgP is also referred to as arnT and pmrK; pmrC is also referred to as eptA). Second, PmrA directly activates expression of the pmrR gene, which encodes an inhibitor of LpxT (12), an enzyme responsible for the incorporation of an additional phosphate group at position 1 of lipid A (Fig. 1A) (14). Modification of the lipid A phosphates with l-Ara4N reduces the LPS negative charge more than does modification of the lipid A phosphates with pEtN. The lipid A phosphates are modified with both l-Ara4N and pEtN in Salmonella harvested from macrophages (15).
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Fig. 1 Signal-dependent control of lipid A modifications.
(A) Schematic representation of the lipid A moiety of the LPS and the lipid A modifications that alter its negative charge. In Salmonella, the predominant lipid A species is hexa-acylated and phosphorylated at the 1 and 4′ positions. LpxT adds a second phosphate group to the 1 position, resulting in a 1-diphosphorylated (1-PP) species. Alternatively, the lipid A 1-phosphate can be modified with pEtN by PmrC. The 4′-phosphate can be modified with l-Ara4N by Ugd and PbgP. Alternatively, but to a lesser extent, the 4′-phosphate may be modified by the addition of pEtN and the 1-phosphate modified by the addition of l-Ara4N. PmrR binds to LpxT, inhibiting its activity. (B) Model for activation of the PhoP/PhoQ and PmrA/PmrB two-component systems and regulation by the PhoP and PmrA proteins. Transcription of PhoP-activated genes is promoted when the sensor PhoQ experiences low cytoplasmic pH (31), low periplasmic Mg2+, or the presence of particular antimicrobial peptides in the periplasm. Transcription of PmrA-activated genes is promoted when the sensor PmrB experiences Fe3+, Al3+, or a mildly acidic pH in the periplasm (4). PmrA is also activated in low Mg2+ by the PhoP-activated pmrD gene product. Phosphorylated PhoP promotes transcription of the pmrD and lpxT genes. Phosphorylated PmrA promotes transcription of the pbgP, pmrC, pmrR, and ugd genes. The pbgP, ugd, and pmrC genes encode enzymes that compete with LpxT for the modification of the lipid A 1-phosphate. The lipid A modifications catalyzed by PbgP, Ugd, and PmrC reduce the negative charge of lipid A. (C) Schematic of the lipid A structure over time after bacteria encounter low Mg2+ or a mildly acidic pH. Under low Mg2+, lpxT is activated by PhoP within 5 min, resulting in 1-PP lipid A. The additional phosphate group at the 1-phosphate prevents the addition of pEtN at this position. By 30 min, lipid A is preferentially modified at the 4′-phosphate with l-Ara4N. PmrR later inhibits LpxT, thereby preventing the incorporation of a phosphate at the 1 position. pEtN is not detected at later times despite inhibition of LpxT by PmrR, suggesting that l-Ara4N–modified lipid A is not a good substrate for PmrC. In a mildly acidic pH, transcription of lpxT gene occurs after that of genes encoding Ugd, PbgP, PmrC, and PmrR, resulting in lipid A modified with both l-Ara4N and pEtN.
PmrA is a response regulator that functions with the sensor PmrB as a two-component system. PmrA is activated (phosphorylated) when its cognate sensor PmrB detects Fe3+ (16) or a mildly acidic pH (17) in the periplasm (Fig. 1B) or when the noncognate sensor PhoQ is activated by its specific signals such as low periplasmic Mg2+ (18, 19). The latter activation entails PhoQ-mediated phosphorylation of its cognate response regulator PhoP (20), transcription of the PhoP-activated gene pmrD (18), and protection of phosphorylated PmrA by the PmrD protein (Fig. 1B) (21). Therefore, transcription of PmrA-activated genes is faster when the inducing signal is Fe3+ or a mildly acidic pH than when the inducing signal is low Mg2+ (Fig. 1, B and C) (22).
Modification of the lipid A phosphates with l-Ara4N and pEtN confers resistance to the cationic peptide antibiotic polymyxin B by reducing the negative charge of the lipid A (7, 13). A pmrA null mutant is 10,000 times more sensitive to polymyxin B than is wild-type Salmonella (16). Polymyxins constitute a "last-resort" antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae (23) but are not used to treat Salmonella infections. However, the better understanding of the PmrA/PmrB and PhoP/PhoQ two-component systems uncovered in Salmonella and Escherichia coli (4, 24) has provided a framework to understand the molecular basis for resistance to polymyxins in clinical isolates of other species where these systems have not been as thoroughly investigated (25).
Here, we provide a molecular explanation for how wild-type Salmonella decorates its lipid A phosphates with different chemicals depending on the signal that activates the PhoP and PmrA proteins (16–18). We establish that the different lipid A profiles reflect the distinct expression kinetics of the genes that encode lipid A–modifying proteins when the inducing signals act on PmrB versus PhoQ. Unexpectedly, the strongest reduction in lipid A negative charge requires a prior increase in its negative charge. Our data also suggest that environmental signals control a broad array of bacterial cellular structures by altering the abundance of undecaprenyl phosphate, an essential lipid molecule that transports sugar substrates for incorporation into various cell envelope structures.
PhoP promotes transcription of the lpxT gene in low Mg2+
The PhoP/PhoQ and PmrA/PmrB two-component systems are the major activators of lipid A modifications in Gram-negative bacteria (2). Lipid A modifications that increase the resistance of Salmonella to polymyxin B reduce the negative charge of lipid A; however, all the lipid A–modifying proteins encoded by genes that are activated by PhoP or PmrA, or both, that have been described in the literature either reduce or do not alter the negative charge of lipid A (2). Therefore, we hypothesized that PhoP or PmrA, or both, may repress lpxT expression because the LpxT protein increases lipid A's negative charge (14). Unexpectedly, we found that the abundance of lpxT mRNA in wild-type Salmonella was 10 times higher after growth in low Mg2+ (10 μM) than in high Mg2+ (10 mM) (Fig. 2A), which are inducing and repressing conditions, respectively, for both the PhoP/PhoQ (24) and PmrA/PmrB systems (Fig. 1B) (4). In agreement with the mRNA data, the abundance of the LpxT protein also increased in low Mg2+ compared to high Mg2+ (fig. S1). Transcriptional activation of the lpxT gene in low Mg2+ was not observed in a phoP single mutant or a phoP pmrA double mutant; by contrast, it was normal in a pmrA single mutant (Fig. 2A). These results indicate that low Mg2+ promoted lpxT transcription in a phoP-dependent but pmrA-independent manner.
Fig. 2 PhoP promotes lpxT transcription directly by binding to the lpxT promoter.
(A) Quantification of lpxT transcripts by quantitative polymerase chain reaction (qPCR) before and 60 min after switching wild-type (WT; 14028s) and pmrA (EG7139), phoP (MS7953s), and phoP pmrA (EG12443) mutant Salmonella from high-Mg2+ medium (10 mM MgCl2) to low-Mg2+ medium (10 μM MgCl2) at pH 7.7, normalized to the rrs (16S ribosomal RNA) transcript. (B) Genomic structure and partial nucleotide sequence of the Salmonella yeiR-lpxT chromosomal region. The sequence in red corresponds to the PhoP box in the lpxT promoter, the underlined sequence to the yeiR stop codon, +1 to the lpxT transcription start site (TSS), and lowercase sequence (atg) to the lpxT start codon. (C) Primer extension analysis of lpxT in WT (14028s) and phoP mutant (MS7953s) Salmonella grown in N-minimal medium (pH 7.7) containing high Mg2+ or low Mg2+. Lanes C, G, A, and T indicate the sequence of the lpxT promoter. The boxed "G" indicates the TSS of lpxT. (D) Deoxyribonuclease I (DNase I) footprinting analysis of the lpxT promoter region using the indicated amounts of purified phosphorylated PhoP-6×His protein. Lanes A, T, C, and G indicate the sequence of the lpxT promoter. The sequence highlighted in red corresponds to the PhoP binding site. (E) Filter assay for binding of the purified PhoP protein to the WT lpxT promoter and to the mutant lpxTB promoter, which contains substitutions in the PhoP binding site. The sequence of the WT lpxT promoter and the nucleotide substitutions in the mutant lpxTB promoter are shown in the box above the graph. The sequence highlighted in red corresponds to the PhoP binding site. The graph shows the percentage of 32P signal compared to the maximum for binding of the indicated amount of PhoP protein to 32P-labeled WT and promoter fragments. Data are representative of three independent experiments. (F) Abundance of lpxT transcript before and 60 min after switching WT (14028s), phoP (MS7953s), and lpxTB (XH16) Salmonella from N-minimal medium (pH 7.7) containing high Mg2+ to low Mg2+, normalized to the rrs transcript. Mean and SD of three independent experiments are shown.
PhoP binding to the lpxT promoter is required for lpxT transcriptional activation
According to Salmonella genome annotations, the lpxT start codon is located 35 nucleotides (nt) downstream of the yeiR stop codon (Fig. 2B). The short intergenic distance between yeiR and lpxT suggested that these genes form an operon and that PhoP promotes transcription of both yeiR and lpxT. However, yeiR mRNA abundance was not altered upon a switch from high to low Mg2+ medium (fig. S2). These results argue that separate promoters transcribe the Salmonella yeiR and lpxT genes under PhoP-inducing conditions, as proposed for the E. coli homologs of these genes (10).
We mapped the lpxT TSS to a G residue 25 nt upstream of the lpxT start codon in wild-type Salmonella grown in low Mg2+ (Fig. 2C). No transcript was detected in cultures of wild-type Salmonella grown in high Mg2+ or in a phoP mutant grown in low Mg2+ (Fig. 2C). These results agree with the lpxT expression behavior of a different Salmonella strain grown under a different PhoP-inducing condition (26). Moreover, they support the notion that wild-type Salmonella activates lpxT transcription during growth in low Mg2+ in a phoP-dependent manner and independently of transcription of the yeiR gene.
We identified a putative PhoP binding site within the yeiR coding region (Fig. 2B). This site is a bona fide PhoP binding site because DNase I footprinting analysis demonstrated that phosphorylated PhoP, which is the active form of the PhoP protein (27), protects nucleotides −47 to −19 relative to the TSS, overlapping the predicted PhoP binding site (Fig. 2D) (28). In agreement with the notion that PhoP activates lpxT transcription directly (that is, by binding to the lpxT promoter), substitution of 3 nt of the PhoP binding site markedly decreased binding of the PhoP protein to a DNA fragment harboring the lpxT promoter (Fig. 2E). Furthermore, an engineered strain with the 3-nt substitutions in the chromosomal copy of the PhoP binding site in the lpxT promoter failed to increase the abundance of lpxT mRNA in low Mg2+ (Fig. 2F), behaving like the phoP null mutant (Fig. 2F). A separate engineered strain with a different 3-nt substitution in the PhoP binding site of the lpxT promoter (lpxTA) also failed to increase lpxT mRNA amounts in low Mg2+ (fig. S3).
Collectively, the results described in this section establish that PhoP directly promotes lpxT transcription when Salmonella experiences low Mg2+. This result is paradoxical because low Mg2+ also promotes expression of the pmrR gene, which encodes an LpxT inhibitor, through the stabilization of the PmrA protein by the PhoP transcriptional target PmrD (Fig. 1B) (12). Next, we explored the phenotypic consequences of PhoP promoting the transcription of both lpxT and pmrR by examining the kinetics with which the mRNAs corresponding to lipid A phosphate–modifying genes are produced, as well as their impact on the resulting lipid A profiles.
The lpxT gene is transcribed before other lipid A phosphate–modifying genes when the inducing signal is low Mg2+
We analyzed the abundance of lpxT, pbgP, pmrC, pmrR, and ugd transcripts at different times after wild-type Salmonella was switched from noninducing (high Mg2+) to inducing (low Mg2+) conditions. The lpxT transcript reached a maximum within 5 min of the shift and then decreased, eventually reaching preinduction values by 120 min (Fig. 3). This behavior differed from the expression of the pmrR, pbgP, pmrC, and ugd genes, which peaked between 20 and 60 min and retained >50% of the maximum values at 120 min (Fig. 3).
Fig. 3 The lpxT gene is expressed earlier relative to other lipid A–modifying genes in low Mg2+.
Quantification of the indicated transcripts at different times after switching WT Salmonella (14028s) from high Mg2+ to low Mg2+ at pH 7.7, normalized to the rrs transcript. Data are means ± SD of three independent experiments.
We reasoned that lpxT transcription precedes that of pbgP, pmrC, pmrR, and ugd in wild-type Salmonella experiencing low Mg2+ because PhoP activates the lpxT promoter directly (Fig. 2) but acts on the pbgP, pmrC, pmrR, and ugd genes indirectly by increasing transcription from the pmrD promoter, resulting in synthesis of the PmrD protein, the protector of phosphorylated PmrA (Fig. 1B) (18). In support of this notion, pmrD mRNA amounts increased at the same time as those of lpxT, preceding the increase in pbgP, pmrC, pmrR, and ugd mRNAs (Fig. 3). Thus, lpxT is transcribed before other genes that encode proteins that target the same lipid A position and before the LpxT inhibitor–encoding gene pmrR.
PhoP-dependent transcription of lpxT favors lipid A modification with l-Ara4N during growth in low Mg2+
The rapid and transient lpxT expression exhibited by Salmonella experiencing low Mg2+ (Fig. 3) resulted in a rapid and transient presence of two phosphates at position 1 of lipid A (1-PP), the addition of which is catalyzed by LpxT (14). We detected the presence of radiolabeled 1-PP lipid A within 10 min of switching wild-type Salmonella from high to low Mg2+ (Fig. 4A) in the presence of radiolabeled phosphate. The abundance of 1-PP lipid A then decreased, and 1-PP lipid A was no longer observed by 1 hour (fig. S4).
Fig. 4 PhoP-dependent lpxT expression promotes lipid A modification with l-Ara4N in low Mg2+ but not in a mildly acidic pH.
(A) Autoradiogram of a thin-layer chromatography (TLC) plate showing lipid A from WT Salmonella (14028s) at different times after they were switched from N-minimal medium (pH 7.7) containing high Mg2+ to low Mg2+ in the presence of radiolabeled phosphate. Data are representative of three independent experiments. The graph below the autoradiogram shows the quantification of lipid A 1-PP from the image, representing mean and SD (normalized to the total signal of each lane). (B) TLC showing lipid A from WT (14028s), lpxTB (XH16), and lpxT (DC72) Salmonella grown in N-minimal medium (pH 7.7) containing high Mg2+, N-minimal medium (pH 7.7) containing low Mg2+, or N-minimal medium (pH 4.9) containing high Mg2+ in the presence of radiolabeled phosphate. (C) TLC showing lipid A from WT (14028s), lpxT (DC72), pbgP (EG9241), pbgP lpxT (XH246), ugd (EG17898), and ugd lpxT (XH251) Salmonella grown in N-minimal medium (pH 7.7) containing high Mg2+ or low Mg2+. Data are representative of two independent experiments. The 1-PP band shows different mobility in each of the images because the samples in each panel were run for different amounts of time. Asterisks (*) indicate positions of lipid A containing l-Ara4N, and open circles (o) indicate positions of lipid A containing pEtN.
The l-Ara4N modification was first detected at 20 min after the switch to low Mg2+ (Fig. 4A). By 1 hour, the lipid A population contained combinations of l-Ara4N with other modifications including 2-OH myristation or palmitation, but not pEtN (fig. S4). Thus, the switch to low Mg2+ resulted in the transient production of 1-PP lipid A, which decreased in abundance and then disappeared as the l-Ara4N modification was detected. These results suggest that the transient increase in 1-PP lipid A taking place shortly after the switch to low Mg2+ favors further modification with l-Ara4N over modification with pEtN.
If the transient increase in 1-PP lipid A mediated by LpxT is required for the subsequent lipid A decoration with l-Ara4N and no pEtN, an lpxT mutant should have both l-Ara4N and pEtN in its lipid A. As predicted, the lipid A of the lpxT mutant was undermodified with the l-Ara4N but hypermodified with various double modifications of l-Ara4N and pEtN (Fig. 4B). The lipid A identities were verified by mass spectrometry (table S1). Moreover, a pbgP single mutant had little pEtN in its lipid A (Fig. 4C) despite its inability to produce l-Ara4N (8). By contrast, pEtN was the dominant modification in the lipid A of the isogenic pbgP lpxT double mutant (Fig. 4C), reinforcing the notion that the addition of a phosphate group to lipid A by LpxT hinders the incorporation of pEtN into lipid A even in organisms that cannot make l-Ara4N. In agreement with this notion, wild-type and pmrC mutant Salmonella exhibited similar lipid A profiles during growth in low Mg2+ (fig. S5). Furthermore, disruption of both pmrA and lpxT abolished all l-Ara4N, pEtN, and 1-PP modifications (fig. S6). Cumulatively, these results indicate that LpxT promotes modification of lipid A with a single l-Ara4N over pEtN or double modification with both l-Ara4N and pEtN.
Because the lpxT gene is transcribed even in a phoP mutant (Fig. 2A), albeit to a lesser extent than in wild-type Salmonella, we wondered whether the increased lpxT transcription promoted in low Mg2+ by the PhoP protein was responsible for the l-Ara4N–modified lipid A (Fig. 4). Thus, we examined the lipid A profile of the lpxT promoter mutant (lpxTB) that is refractory to PhoP activation (Fig. 2F). The use of this mutant avoids potentially confusing effects resulting from the pleiotropic effects of phoP inactivation, such as the inability to activate the PmrA protein in low Mg2+ (Fig. 1B) (18).
The lpxTB promoter mutant harbored less lipid A species with l-Ara4N and more lipid A species doubly modified with l-Ara4N and pEtN compared to the wild-type strain (Fig. 4B). This phenotype is specific to the PhoP-inducing condition of low Mg2+ because the lpxTB mutant retained normal 1-PP modification after growth under the noninducing high Mg2+ condition (Fig. 4B). The latter result indicated that the lpxT gene itself was functioning properly in the lpxT promoter mutant (lpxTB). That is, the inability of the lpxT mutant promoter to respond to PhoP did not compromise lpxT expression under noninducing conditions for PhoP. Moreover, when grown in low Mg2+, the pmrC single mutant and the pmrC lpxTB double mutant displayed the same lipid A profile containing l-Ara4N and no pEtN (fig. S5). These results established that PhoP activation of lpxT transcription is required for normal lipid A modification with l-Ara4N.
Similar transcription timing of lpxT and other lipid A modification genes results in lipid A with pEtN during growth in a mildly acidic pH
A mildly acidic pH activates both PmrB and PhoQ, resulting in transcription of PmrA- and PhoP-activated genes, respectively (Fig. 1B) (17, 29, 30). However, these sensors differ in that PmrB responds to a mildly acidic pH through protonation of periplasmic histidine residues (17), whereas PhoQ does so through its cytoplasmic region through a mechanism that does not involve histidine residues (31). In addition, full activation of the PhoP/PhoQ system under a mildly acidic pH requires the PhoP-activated gene ugtL, which encodes a protein that stimulates PhoQ autophosphorylation (30).
We reasoned that the kinetics with which a mildly acidic pH induces PmrA- and PhoP-activated genes might differ from that taking place in low Mg2+ because low Mg2+ activation requires the PhoP-dependent transcription of the pmrD gene (18), which specifies a protein that protects phosphorylated PmrA from dephosphorylation by its cognate sensor PmrB (21), and results in transcription of genes directly activated by PmrA. By contrast, PmrB-mediated activation of PmrA by a mildly acidic pH does not require PhoP or PmrD (Fig. 1B). That is, when the inducing signal is a mildly acidic pH, the PhoP-dependent lpxT transcription may not precede that of PmrA-activated genes, as observed in low Mg2+.
When wild-type Salmonella was shifted from noninducing conditions to medium with a mildly acidic pH, the mRNA amounts of the PmrA-activated genes pbgP, pmrC, and pmrR reached a maximum within 5 min (Fig. 5A). By contrast, lpxT mRNA amounts reached a maximum at 10 min after the shift (Fig. 5A), raising the possibility that PmrR may be available to readily inhibit LpxT activity when the inducing condition is a mildly acidic pH. If true, the lipid A profile of wild-type Salmonella grown in a mildly acidic pH should no longer display the lpxT-dependent accumulation of l-Ara4N observed when the inducing signal was low Mg2+ (Fig. 4). As predicted, wild-type Salmonella, the lpxT promoter mutant, and the lpxT deletion mutant produced identical lipid A profiles with an abundance of pEtN-modified lipid A when grown in a mildly acidic pH (Fig. 4B). This result indicates that LpxT is not actively adding phosphate to lipid A when the inducing signal is a mildly acidic pH, resulting in a lipid A modified with both pEtN and l-Ara4N.
Fig. 5 Effects of low Mg2+, acidic pH, and PhoP-dependent transcription of the lpxT gene on polymyxin B resistance.
(A) Quantification of the indicated transcripts at different times after switching WT Salmonella (14028s) from high Mg2+ at pH 7.7 to high Mg2+ at pH 4.9, normalized to the rrs transcript. Data are means ± SD of three independent experiments. (B) Percent survival of WT (14028s), pmrA (EG7139), lpxTB (XH16), and lpxT (DC72) Salmonella exposed to polymyxin B after growth in N-minimal medium (pH 7.7) with low Mg2+ or that (pH 4.9) with high Mg2+ (acidic pH). Survival values were calculated as relative to the original inoculum. Mean and SD of three independent experiments are shown. Unpaired Student's t tests were performed comparing mutant strains with the WT under the same growth condition. *P < 0.05, **P < 0.01, and ****P < 0.0001; n.s. indicates P > 0.05 (not significant).
A mildly acidic pH promotes greater polymyxin B resistance than does low Mg2+
Genetic experiments previously revealed that Salmonella mutants defective in the pbgP or ugd genes, which fail to modify lipid A with l-Ara4N, are 100 times more sensitive to polymyxin B than are wild-type Salmonella after growth in low Mg2+ (9). By contrast, a pmrC mutant, which is defective in lipid A modification with pEtN, displayed marginally increased polymyxin B sensitivity compared to wild-type Salmonella (9). We have recapitulated these results (fig. S7) and determined that the lpxT null mutant was slightly more resistant to polymyxin B than was wild-type Salmonella (fig. S7).
We hypothesized that growth in low Mg2+ enhanced polymyxin B resistance compared to growth in a mildly acidic pH because the lipid A of wild-type Salmonella grown in low Mg2+ was modified with l-Ara4N and no pEtN (Fig. 4B) and also because inactivation of the genes required for lipid A modification with l-Ara4N decrease polymyxin B resistance much more than does inactivation of the pmrC gene (fig. S7). Unexpectedly, survival of wild-type Salmonella exposed to polymxyin B was seven times higher after growth in a mildly acidic pH than it was in low Mg2+ (Fig. 5B). As predicted from the results presented in the previous section (Fig. 4, B and C), the lpxT promoter mutant and the lpxT null mutant exhibited similar polymyxin B resistance as did the wild-type strain when the inducing condition was a mildly acidic pH (Fig. 5B). By contrast, the lpxT promoter mutant was more resistant to polymxyin B than was the wild-type strain, and the lpxT null mutant was more resistant than the lpxT promoter mutant when the inducing condition was low Mg2+ (Fig. 5B). Control experiments demonstrated that the pmrA mutant is several orders of magnitude more sensitive than the wild-type strain regardless of the inducing condition (Fig. 5B), in agreement with previous results (18). In summary, Salmonella survival to polymxyin B is higher when its lipid A is modified with both l-Ara4N and pEtN compared to modification with only l-Ara4N (Fig. 6).
Fig. 6 PhoP-dependent transcriptional activation of the lpxT gene is conserved in a subset of Gram-negative species.
(A) Alignment of the nucleotide sequence of the lpxT promoter region in selected enteric bacterial species reveals conservation of the PhoP binding site (underlined). The boxed "G" indicates the transcription start site of lpxT mapped in Salmonella. Asterisks (*) denote positions that are conserved in all four species: STM, S. enterica serovar Typhimurium; ECO, E. coli; CFD, C. freundii; KPN, K. pneumoniae. (B) Abundance of lpxT mRNA before and 60 min after WT (MG1655) and phoP (EG12976) E. coli were switched from N-minimal medium (pH 7.7) containing high Mg2+ to low Mg2+, normalized to the rrs transcript. Mean and SD of three independent experiments are shown.
PhoP-dependent activation of lpxT is conserved in a subset of Gram-negative species
A wide range of Gram-negative species have the ability to modify their lipid A phosphates. However, the specific chemical modification and the regulation of the genes responsible for such modifications vary among species. For example, Yersinia pestis differs from Salmonella in lacking a pmrC gene and in its pbgP promoter being directly activated by both the PhoP and PmrA proteins (32). In P. aeruginosa, the LpxT enzyme can transfer an additional phosphate to lipid A (33), and the pmrC gene is activated by the ColR/ColS two-component system but not by PmrA/PmrB (34).
To determine whether the PhoP-mediated activation of lpxT transcription identified in Salmonella (Fig. 2) is conserved in other Gram-negative species, we examined bacterial genomes for sequences related to the Salmonella lpxT, pbgP, phoP, and pmrC genes, as well as for sequences resembling a PhoP binding site in the lpxT promoter (table S2). Citrobacter freundii, K. pneumoniae, and E. coli harbor a predicted PhoP binding site upstream of the lpxT coding region, which is highly conserved with the one in the Salmonella lpxT promoter (Fig. 6A). This suggests that lpxT transcription is promoted in a phoP-dependent manner in these species as well. In addition, a 10-nt stretch located four bases upstream of the lpxT TSS is fully conserved in the four species, suggesting that it is the binding site for an as-yet-unidentified regulatory molecule that controls lpxT expression. By contrast, Yersinia and Pseudomonas, species in which the pmrC gene is either absent or not regulated by the PmrA/PmrB system, lack sequences resembling a PhoP binding site in their lpxT promoter regions. This analysis suggests a correlation between PhoP activation of lpxT transcription and the presence of a PmrA-activated pmrC gene.
We determined that lpxT mRNA abundances were threefold higher in wild-type E. coli grown in low Mg2+ than in those grown in high Mg2+ (Fig. 6B). By contrast, an E. coli phoP null mutant displayed similarly low amounts of lpxT mRNA under both inducing and repressing conditions (Fig. 6B). Although the expression behavior of the E. coli lpxT gene was qualitatively similar to that of the Salmonella lpxT homolog, the threefold induction exhibited by the E. coli lpxT gene was much lower (Fig. 6B) than the 10-fold increase exhibited by the Salmonella lpxT gene (Fig. 2A) under identical inducing conditions. Given that the E. coli PmrD protein is defective in activating the PmrA protein (35), the lower induction of the lpxT gene of E. coli might reflect the decreased need to activate the lpxT gene ahead of pmrR when the inducing condition is low Mg2+.
We have established that differences in the expression kinetics of lipid A–modifying genes are responsible for the distinct lipid A profiles exhibited by Salmonella experiencing different inducing conditions for these genes. That is, both low Mg2+ and a mildly acidic pH activate the PhoP/PhoQ and PmrA/PmrB systems, causing transcription of their target genes (Figs. 2 and 5). However, these two inducing conditions give rise to different lipid A modifications because transcription of the lpxT gene precedes that of other lipid A modification genes including pbgP, pmrC, and ugd, as well as pmrR, which encodes an inhibitor of LpxT when the inducing signal is low Mg2+ but not when it is a mildly acidic pH (Figs. 2 and 5). PhoP-activated lpxT transcription is necessary for lipid A modification with l-Ara4N and no pEtN because preventing this activation results in a lipid A modified with both l-Ara4N and pEtN (Fig. 4B). Given that LpxT is responsible for the incorporation of an additional phosphate into lipid A (14, 36), our results indicate that, paradoxically, a decrease in lipid A negative charge resulting from covalent lipid A modification with l-Ara4N requires a previous lipid A modification that increases its negative charge.
The expression kinetics of lipid A phosphate modification genes determines the distinct chemical modifications of the lipid A phosphates
When Salmonella experiences low Mg2+, the PhoP/PhoQ system is activated before the PmrA/PmrB system because, under this inducing condition, PmrA activation is dependent on the PhoP-activated pmrD gene (Fig. 1B) (18). By being directly activated by the PhoP protein (Fig. 2), lpxT is expressed before the lipid A–modifying genes that are directly controlled by the PmrA protein (Fig. 3), most critically pmrR, which specifies an LpxT inhibitor (12). Therefore, PhoP-dependent lpxT expression induced in low Mg2+ results in 1-PP lipid A (Fig. 4A), which favors further modification of lipid A phosphates with l-Ara4N and the exclusion of pEtN (Fig. 4B).
By contrast, when the activation signal is a mildly acidic pH, PmrA/PmrB activation does not lag behind that of PhoP/PhoQ (Fig. 1B). This is because PmrB detects a mildly acidic pH in the periplasm (17) and does not depend on the PmrD protein to activate PmrA. In addition, PhoQ responds to mild acidification through its cytoplasmic domain (31). Thus, the early pmrR expression taking place under a mildly acidic pH, which results in the production of the PmrR protein, is anticipated to decrease LpxT activity, resulting in a lipid A with both l-Ara4N and pEtN modifications (Fig. 4B). That is, the lpxT gene is expressed in a mildly acidic pH (Fig. 5A), but the LpxT protein is inhibited by PmrR (12).
In agreement with the model presented above (Fig. 1B), mutants lacking the lpxT gene or with nucleotide substitutions in the lpxT promoter that render it refractory to activation by PhoP behave like wild-type Salmonella in a mildly acidic pH but show different behaviors when the inducing signal is low Mg2+ (Figs. 4B and 5B). Our findings raise the possibility of an analogous mechanism being responsible for lipid A deacylation by the PhoP-activated PagL protein occurring only when modification with l-Ara4N and pEtN is prevented (37).
Transcription kinetics governs critical cellular processes
Our results highlight the fundamental role that gene expression timing plays in physiological responses to stress conditions. Low Mg2+ and a mildly acidic pH promote different lipid A profiles (Fig. 4B) despite stimulating the expression of the same set of lipid A modification genes (Figs. 2 and 5). This is not the only example of expression timing, rather than simply the expression or nonexpression of specific genes at steady-state, dictating phenotypic outcomes. For example, a surge in the transcription of phoP is necessary to jump-start Salmonella's virulence program (20). This surge requires PhoP to positively feedback on its own transcription because a strain constitutively expressing PhoP is attenuated for virulence despite achieving the same steady-state abundance of PhoP-activated transcripts (20). Likewise, the increased amounts of phosphorylated PhoP generated after several hours in low Mg2+ enable Salmonella to transcribe PhoP-dependent genes that require high amounts of active PhoP protein (38). That is, the increased amounts of phosphorylated PhoP in this case depend on the Mg2+ importer MgtA removing Mg2+ away from the periplasmic Mg2+-sensing domain of the sensor PhoQ (38). Because transcription elongation into the mgtA coding regions is triggered in low cytoplasmic Mg2+ (39), expression of a subset of PhoP-activated genes takes place only when the bacterium experiences the signals promoting mgtA expression. Furthermore, the stimulation of PmrB by Fe3+ activates PmrA to different extents at early and late times due to a negative feedback loop that hinders Fe3+ access to PmrB (12). These examples illustrate how cellular behaviors in response to particular environmental conditions change over time even when the condition triggering the response does not change.
Transcript abundance does not always reflect the activity of the corresponding protein
mRNA abundance is often used as proxy for the amounts and activities of the corresponding proteins. This is because mRNA determinations are straightforward across a genome, whereas activity assays are not available for the vast majority of gene products. Our data demonstrate two examples in which the presence of particular mRNAs is not accompanied by the activity of the specified gene products. First, when the lpxT gene was transcribed in response to a mildly acidic pH (Fig. 5A), the LpxT-mediated modification of lipid A was not observed (Fig. 4B) presumably because of the presence of the LpxT inhibitor PmrR (12). Likewise, the pmrC gene was highly induced in low Mg2+ (Fig. 3), but the lipid A did not contain detectable pEtN (Fig. 4B) due to preceding PhoP-dependent lpxT transcription (Fig. 3) favoring the incorporation of l-Ara4N over the incorporation of pEtN (Fig. 4B).
Environmental control of LpxT expression affects multiple pathways
As discussed above, Salmonella favors lipid A modified with l-Ara4N under low Mg2+ and with both l-Ara4N and pEtN when experiencing a mildly acidic pH (Fig. 1C). The fact that the lipid A from Salmonella harvested from inside macrophages contains both l-Ara4N and pEtN (15) is in agreement with three notions: first, that the macrophage phagosome containing Salmonella is mildly acidic (40); second, that an acidic pH is critical to activate the PhoP/PhoQ system inside macrophages (40); and third, that an acidic pH is the signal promoting expression of lipid A phosphate–modifying genes inside macrophages (15).
Wild-type Salmonella grown in a mildly acidic pH is seven times more resistant to polymyxin B than when grown in low Mg2+ (Fig. 5B). Because polymyxins are not used to treat Salmonella infections, our findings raise the possibility of the lipid A modification with both l-Ara4N and pEtN, which is favored under a mildly acidic pH, mediating resistance to host-derived products, decreasing recognition by the host immune system, playing a yet-to-be identified function, or a combination of these roles. We note that a pmrA null mutant, which is unable to modify lipid A with either l-Ara4N or pEtN (2), is hypervirulent in cultured macrophages and in mice inoculated through the intraperitoneal route (41).
Finally, the LpxT protein removes the distal phosphate from its substrate—undecaprenyl diphosphate (C55-PP)—and produces C55-P, which is an essential carrier that shuffles sugar moieties across the inner membrane for various biosynthetic pathways (14, 42). The identification of environmental conditions and regulatory factors that control lpxT gene expression raises the possibility of the essential C55-P metabolism being affected by the environmental conditions encountered by bacteria. This hypothesis is supported by the observation that the PhoP and PmrA proteins have been implicated in controlling the expression of the ybjG gene, which, like lpxT, encodes a C55-PP phosphatase that is required for recycling C55-P (43) in E. coli.
Bacterial strains, plasmids, primers, and growth conditions
S. enterica serovar Typhimurium strains were derived from the wild-type strain 14028s. Unless otherwise stated, bacteria were grown at 37°C in LB broth or in N-minimal medium (pH 7.7) (44) supplemented with 0.1% casamino acids, 38 mM glycerol, and 10 mM or 10 μM of MgCl2. When necessary, antibiotics were added at the following final concentrations: ampicillin (50 μg/ml), chloramphenicol (20 μg/ml), kanamycin (50 μg/ml), and tetracycline (10 μg/ml). P22 transduction of Salmonella strains was performed as described (45). E. coli DH5a was used as a host for the preparation of plasmid DNA. Bacterial strains and plasmids used in this study are listed in table S3. Primers used in this study are listed in tables S4 and S5.
All experiments were carried out with wild-type S. enterica serovar Typhimurium strain 14028s, wild-type E. coli strains MG1655 or W3110, and mutant derivatives. LB (Becton, Dickinson and Co.), Super Optimal Broth (SOB) (Becton, Dickinson and Co.), or Super Optimal broth with Catabolite repression (SOC) (SOB supplemented with 20 mM glucose) media were used for cloning and strain construction, as noted. When necessary, ampicillin was used at 50 μg/ml, and tetracycline was used at 12.5 μg/ml. Fusaric acid (FA) plates were used for selection against tetracycline-resistant bacteria [LB agar (40 g/liter; Becton, Dickinson and Co.), NaH2PO4·H2O (10 g/liter) (Sigma-Aldrich), chlortetracycline hydrochloride (60 mg/liter) (Sigma-Aldrich), FA (12 mg/liter) (Acros Organics), and 0.1 mM ZnCl2 (Sigma-Aldrich)] (46). For experiments in which gene expression was measured, bacteria were grown in N-minimal medium (pH 7.7 or pH 4.9) (47) and the indicated concentration of MgCl2. For time course experiments, cells were grown in N-minimal medium (pH 7.7; 10 mM Mg2+) to log phase and then either washed with fresh N-minimal medium (pH 7.7; no Mg2+) twice and resuspended with N-minimal medium (pH 7.7; 10 μM Mg2+) or resuspended with N-minimal medium (pH 4.9; 10 mM Mg2+) before being incubated for the indicated times. All incubations were carried out at 37°C with shaking at 250 rpm.
Strain construction
Strains harboring chromosomal point mutations in the lpxT promoter region were constructed by a method based on selection for loss of tetracycline resistance (46). First, the promoter region of lpxT was replaced with a tetracycline resistance (TetR) cassette. This was carried out by transforming wild-type Salmonella (14028s) expressing the phage λ Red recombinase machinery from plasmid pKD46 with a DNA fragment containing the TetR cassette from transposon Tn10 flanked by ~60-nt regions of nucleotide sequence identity to the lpxT promoter region. The DNA fragment was generated using primers W3423 and W3424 for strain XH16 and primers W3427 and W3428 for strain XH15, with genomic DNA from MS7953s as a template. Electroporated cells were recovered in SOC medium at 30°C and plated on LB/Ap/Tet/plates and incubated at 30°C. The resulting strains were grown to mid-log phase in SOB/Ap/10 mM l-arabinose at 30°C and made electrocompetent by washing twice in ice-cold water and once in ice-cold 10% glycerol. Next, a DNA oligo corresponding to the lpxT promoter and containing the desired point mutation(s) was generated using two primers W3425 and W3426 that are reverse complement to each other (W3425 and W3426 for XH16 and W3430 and W3433 for XH15). The resulting product was transformed into electrocompetent cells. Electroporated cells were recovered in SOC at 37°C. Transformants were cured of pKD46 by plating on LB at 42°C and tested for loss of Ap and Tet resistance, and mutations were confirmed by sequencing PCR products generated from purified genomic DNA. To construct the strain specifying a C-terminally FLAG-tagged LpxT protein (XH201), the DNA fragment amplified from pKD3 using primers W4309/W4310 was introduced into wild-type Salmonella 14028s harboring plasmid pKD46.
When necessary, P22 phage transduction was used to move selectable markers. XH246 was generated by using a P22 lysate grown on strain EG9241 to infect strain DC74, which is a derivative of strain DC72 with the Km cassette removed by using plasmid pCP20 (48). XH248 was generated by infecting XH16 with a P22 lysate prepared on strain EG9460. XH251 was generated by infecting DC72 with a P22 lysate prepared on strain EG17898.
RNA isolation and complementary DNA synthesis
A bacterial pellet was isolated from 1 ml of liquid culture and placed immediately on dry ice. The pellet was resuspended in 1 ml of 2:1 RNAprotect reagent (Qiagen)/water and incubated at room temperature for 5 min. After centrifugation at 5000 rpm for 10 min, the pellet was stored at −80°C. The sample was resuspended in 100 μl of lysozyme (10 mg/ml; Sigma-Aldrich) and incubated at room temperature for 2 hours with occasional vortexing. Total RNA was isolated using the RNeasy Mini kit (Qiagen) and the recommended on-column DNase I treatment, according to the manufacturer's instructions. For first-strand complementary DNA (cDNA) synthesis, 1 μg of purified RNA in 16 μl of ribonuclease (RNase)–free water was mixed with 4 μl of VILO Master Mix (Invitrogen) and incubated at 25°C for 10 min, 42°C for 60 min, and 85°C for 5 min. The resulting cDNA was diluted by adding 100 μl of water before qRT-PCR.
qRT-PCR
For quantification of cDNA abundance, 2 μl of diluted cDNA was mixed with 7.5 μl of 2× SYBR Green Master Mix (Applied Biosystems) and 5.5 μl of primer mix (each primer diluted to 72.7 μM in water). Reactions were carried out in a 7500 Real-Time PCR machine (Applied Biosystems) using the manufacturer-provided software and settings. Transcript quantities were determined by fitting CT (threshold cycle) values to a standard curve generated with Salmonella genomic DNA.
DNase I footprinting assay
DNase I foot printing assay was conducted as described (41). DNA fragments corresponding to the lpxT region were generated by PCR using primer W3436 and 32P-labeled primer W3435 with genomic DNA from strain 14028s as a template. Unincorporated [γ-32P]–adenosine 5′-triphosphate was removed by using G-50 microcolumns (GE Healthcare). A total of 2 × 104 cpm of labeled probe (~10 fmol), 200 ng of poly(deoxyinosinic-deoxycytidylic) (Sigma-Aldrich), and purified His-tagged PhoP (PhoP-6×His) (49) were mixed with binding buffer [20 mM Hepes (pH 8.0), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, bovine serum albumin (50 μg/ml), and 10% (v/v) glycerol] in a total volume of 20 μl and incubated at room temperature for 20 min. DNase I (Gibco) (0.01 U), 10 mM CaCl2, and 10 mM MgCl2 were added and incubated at room temperature for 3 min. Final concentrations of PhoP protein were 0, 1, 5, 10, and 20 μM. The reaction was stopped by the addition of 100 μl of a phenol chloroform solution, and the aqueous phase was precipitated with ethanol. The precipitate was dissolved in sequence-loading buffer and electrophoresed on a 6% acrylamide/7 M urea gel, together with a sequence ladder generated with the labeled primer by using the T7 Sequenase 2.0 DNA Sequencing kit (Amersham Biosciences), and the gels were dried and autoradiographed.
Filter-binding assay
Determination of binding efficiency of DNAs to the PhoP protein was carried out by the nitrocellulose-binding method (50). 32P-radiolabeled DNAs were incubated with a series of dilutions of the PhoP protein under the same condition used in the electrophoretic mobility shift assay at room temperature for 20 min. Final concentrations of PhoP protein were 0, 1, 2, 5, 10, and 20 μM. The samples were filtered through 0.45-μm nitrocellulose filters (HAWP, Millipore) under vacuum, and the filters were washed with 5 ml of the binding buffer, air-dried, and quantified using PhosphorImager (GE Healthcare).
Primer extension assay
Cultures were grown as indicated in the figure legend. Total RNA was extracted as described (51). cDNA synthesis was performed using 2 pmol of 32P-labeled 14997 primer with 10 μg of total RNA and 1 U of SuperScript II RNase H2 reverse transcriptase (Life Technologies). The extension products were analyzed by electrophoresis on a 6% acrylamide/7 M urea gel and compared with sequence ladders initiated with the same 32P-labeled primer that was used for primer extension.
Polymyxin B susceptibility assay
Antimicrobial peptide susceptibility assays were conducted as described (7), with modifications. Bacteria were grown in N-minimal medium with 10 μM Mg2+ (pH 7.7) or with 10 mM Mg2+ (pH 4.9) for 4 hours. Bacterial cells were then diluted 200× in phosphate-buffered saline (PBS), combined with 1:1 ratio with polymyxin B (final concentration of 5 μg/ml). After 1-hour incubation at 37°C with aeration, samples were serially diluted in PBS and plated on LB agar plates for enumeration. The percentage survival was calculated as follows: survival (%) = colony-forming units (CFU) of peptide-treated culture/CFU of no-peptide culture × 100.
Isolation of labeled lipid A
Cultures were diluted to an OD600 (optical density at 600 nm) of 0.05 in 5 ml of fresh medium and labeled with 32Pi (2.5 μCi/ml; Amersham Biosciences) in N-minimal medium, as indicated. For the time course analysis, cells were first grown in 5 ml of N-minimal containing 10 mM Mg2+ to log phase, washed twice with N-minimal medium, and resuspended with 5 ml of N-minimal medium containing 10 μM Mg2+ and 32Pi (2.5 μCi/ml) for the indicated time. Cells were harvested by centrifugation, and the isolation of 32P-labeled lipid A was carried out by mild acid hydrolysis, as previously described (8, 52). 32P-lipid A species (~1000 cpm·per lane) were analyzed by TLC in a solvent system of chloroform, pyridine, 88% formic acid, and water (50:50:16:5, v/v) and visualized using a PhosphorImager (GE Healthcare).
Analysis of lipid A by mass spectrometry
Bacterial strains were grown as described earlier in 25 ml of medium for 4 hours. Lipid A was purified according to published protocols (53) and analyzed by the Keck Mass Spectrometry & Proteomics Resource at Yale University.
Western blot assay
Cells were grown as described above. Crude extracts were prepared in B-PER reagent (Pierce) with lysozyme (100 μg/ml) and EDTA-free protease inhibitor (Roche). Samples were loaded onto 4 to 12% NuPAGE gels (Life Technologies) and transferred to nitrocellulose membrane using the iBot machine (Life Technologies). Membranes were blocked with 3% skim milk solution at room temperature for 1 hour. Then, samples were analyzed using anti-FLAG or anti-GroEL antibodies. Rabbit anti-FLAG antibodies were used at 1:4000 dilution. Mouse anti-GroEL antibodies were used as control at 1:12,000 dilution. Secondary horseradish peroxidase–conjugated anti-rabbit or anti-mouse antiserum (GE Healthcare) was used at 1:4000 dilution. Blots were developed with the Amersham ECL Western Blotting Detection Reagents (GE Healthcare) or SuperSignal West Femto Chemiluminescent system (Pierce).
www.sciencesignaling.org/cgi/content/full/11/529/eaar7921/DC1
Fig. S1. LpxT protein amounts in wild-type Salmonella.
Fig. S2. yeiR and lpxT transcript abundances in wild-type Salmonella in low Mg2+.
Fig. S3. Abundance of lpxT transcript in wild-type, phoP, and lpxT promoter mutant Salmonella.
Fig. S4. Lipid A profiles of wild-type Salmonella in low Mg2+.
Fig. S5. Lipid A profiles of wild-type, lpxT promoter, pmrC, and double pmrC lpxT promoter mutant Salmonella.
Fig. S6. Lipid A profiles of wild-type, lpxT, pmrA, and pmrA lpxT Salmonella.
Fig. S7. Resistance of wild-type, pmrA, pbgP, pmrC, and lpxT Salmonella to polymyxin B.
Table S1. Lipid A profiles of isogenic Salmonella strains determined by mass spectrometry.
Table S2. Coexistence of lipid A–modifying genes in the genomes of seven Gram-negative bacterial species.
Table S3. Bacterial strains and plasmids used in this study.
Table S4. Primers used in the construction of strains and plasmids.
Table S5. Primers used in this study for the quantification of transcripts in Salmonella.
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H. Barreteau,
T. Touze
, Deciphering the metabolism of undecaprenyl-phosphate: The bacterial cell-wall unit carrier at the membrane frontier. Microb. Drug Resist. 20, 199–214 (2014).
M. D. Snavely,
J. B. Florer,
C. G. Miller,
M. E. Maguire
, Magnesium transport in Salmonella typhimurium: Expression of cloned genes for three distinct Mg2+ transport systems. J. Bacteriol. 171, 4752–4760 (1989).
T. Watanabe,
Y. Ogata,
R. K. Chan,
D. Botstein
, Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium: I. Transduction of R factor 222 by phage P22. Virology 50, 874–882 (1972).
B. R. Bochner,
H. C. Huang,
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, Positive selection for loss of tetracycline resistance. J. Bacteriol. 143, 926–933 (1980).
, The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase. J. Biol. Chem. 266, 815–823 (1991).
K. A. Datsenko,
B. L. Wanner
, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640–6645 (2000).
, Acetyl phosphate-dependent activation of a mutant PhoP response regulator that functions independently of its cognate sensor kinase. J. Mol. Biol. 300, 291–305 (2000).
J. Carey,
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, Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry 22, 2601–2610 (1983).
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, Phosphorylated PmrA interacts with the promoter region of ugd in Salmonella enterica serovar typhimurium. J. Bacteriol. 182, 3874–3876 (2000).
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, Lipid A modifications characteristic of Salmonella Typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-l-arabinose, phosphoethanolamine and palmitate. Biochemistry 274, 18503–18514 (1999).
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, Isolation and chemical characterization of lipid A from gram-negative bacteria. J. Vis. Exp. e50623 (2013).
Acknowledgments: We thank M. A. Valvano for providing feedback on the manuscript. Funding: This research was supported by NIH grant R01 AI120558 to E.A.G. Author contributions: X.H. and E.A.G. designed the research. X.H. and H.D.C. performed the research. X.H. and E.A.G. analyzed the data. X.H. and E.A.G. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The strains reported in this paper are available to qualified investigators. The mass spectrometry data are available at the University of California, San Diego Metabolomics Workbench (accession no. xhong_20180320_135609; www.metabolomicsworkbench.org).
You are going to email the following Gene expression kinetics governs stimulus-specific decoration of the Salmonella outer membrane
By Xinyu Hong, H. Deborah Chen, Eduardo A. Groisman
Science Signaling 08 May 2018
Activation of the same genes with different kinetics elicits distinct modifications of the Salmonella outer membrane.


text:
Russian Minister Against Probe Into Uzbek Violence
9 June 2005 -- Russian Defense Minister Sergei Ivanov said today his country would not support calls for an international investigation into the violence in Uzbekistan's eastern city of Andijon last month
He said any investigation into the Andijon violence should be conducted by prosecutors and other authorities in Uzbekistan.
Uzbek troops opened fire on protesters on 13 May. The Uzbek government says 173 people were killed but international rights groups and local opposition political parties say the number could be as high as 1,000.

redpajama_set_name: RedPajamaCommonCrawl


ZeroPollution© is the first oral branded ingredient for pollution-induced skin damaged. It is totally natural and can be used both orally and topically. It is the result of several years of research led by Dr. Nuria Caturla.
Besides Zeropollution©, in this edition of Hi Japan we have presented the digitalization concept. We offer to the companies the possibility of making use of a mobile App related to our branded ingredients to show to their consumers how the product is working on them and start a conversation with them. We believe innovation is key for differentiation.
Monteloeder's products were prominently showed in Willfarm's booth. From here we want to congratulate our distributor Willfarm for their differentiated and nice designed booth.
Miss Spain 2010 Paula Guillo, Nutroxsun's ambassador, also was present in this event. She commented that "I share the values of Nutroxsun because is natural, Mediterranean and help your skin to be protected from the damage caused by an excess of sun exposure".
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Home > News > Part Nationalisation of Tata Steel
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Part Nationalisation of Tata Steel
The government has announced that it is willing to take a minority stake in Tata Steel's UK operations in order to attract a buyer for the business.
If a buyer cannot be found in the near future, Indian steel giant Tata may close down its remaining UK operations, costing tens of thousands of jobs. This has prompted the UK government to announce support for potential new buyers (1).
Business secretary, Sajid Javid, announced a new package yesterday entailing support amounting to hundreds of millions of pounds, including loans, grants and a possible equity stake (2).
UK and Welsh governments jointly declared that they would consider an equity stake of up to 25% in the steel plants (2).
The UK operations are currently losing £1m per day and Tata has stated that it wants to sell up "in a time bounded way" (3).
Tata have stated that the disposal of its UK operations is an exercise in exposure reduction (4), illustrated by the sale of the Scunthorpe site to Greybull Capital for a nominal £1 (3).
Two possible buyers have emerged, the first a management buyout backed by the chief of Tata's Port Talbot factory. Steel company Liberty House have also expressed interest in buying parts of the business. (3).
http://www.bbc.co.uk/news/business-36102835
http://www.theguardian.com/business/2016/apr/21/government-take-stake-tata-steel-british-plants
http://www.bloomberg.com/news/articles/2016-03-29/tata-steel-to-study-sale-of-u-k-unit-after-strategic-review
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Happy Bizarre Holidays Month!For most of us August brings the end of summer and a new school year. It's probably a good thing that this month is empty of any National holidays! As parents, we are preparing our children for the new school year with shopping, reviewing and squeezing in those last FUN summer activities. Some of you might even be finally embarking on your summer vacation.
With all of you being very busy, I figured I could bring you a short break with some unique information. There are 48 bizarre and unique holidays celebrated during the month of August. Yep, you read that correct, 48!!! These fun celebrations range from National Watermelon Day to Kiss and Make Up Day to Middle Child's Day. But today is Book Lover's Day! What luck! I sat down to send you an e-mail and found it to be one of my favorite hobby's is celebrated today!
Book reading is an important hobby! Reading is educational, informative, and relaxing. Employer's look for it to be listed on resumes. Reading makes us smarter and happier people. So join me today! Grab a book, find a quiet cozy place, and crack open the cover.


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Turkey – Syria - Q&A - Excerpts from the daily press briefing (18.10.18)
Q - Turkey has continued to launch airstrikes in recent days against northeastern Syria, despite your calls for restraint. How do you plan to convince the Turks?
A - We reiterate our deep concern following recent Turkish airstrikes in northeastern Syria, which left several dead. Once again we call on Turkey and all involved parties to show the utmost restraint and we support efforts to deescalate the situation, in particular those undertaken by the United States.
With Daesh engaging in a large-scale counter-offensive in northeastern Syria, we stress the priority of pursuing the campaign against that terrorist organization. The Syrian Democratic Forces are making a decisive contribution to this fight, with the support of the international coalition against Daesh.
As emphasized by the heads of state and government of France, Germany, Russia and Turkey at the Istanbul summit, there can be no military solution to the Syrian conflict. Only a political solution consistent with UN Security Council Resolution 2254 will provide the means to stabilize Syria in a lasting manner and to guarantee its integrity and the security of its neighbors.
France / Turkey (in French)
Embassy of France in Turkey
Embassy of Turkey in France
France and Turkey
Turkey and the European Union
Turkey / Terrorist fighters - Q&A - From the press briefing (9 Dec. 2019)
Press release - Turkey (29 Nov. 2019)
Turkey – Terrorist fighters - Q&A - From the press briefing (12 Nov. 19)
Syria – Turkey - Q&A - From the press briefing (14 Oct. 19)
Turkey - Q&A - From the Press Briefing (14 Oct. 19)
Turkey – Northeastern Syria (07 Oct. 19)
Turkey - Q&A - Excerpts from the daily press briefing (14.06.19)


If you don't have a Raspberry Pi 3, you should really get one. They are super cool and relatively inexpensive. In this exercise, you will configure a Raspberry Pi 3 to run as a Wireless Access Point, granting connected users access to the Internet. This exercise is great because you will install Kali to the Raspberry Pi, edit files, change file permissions, configure network interfaces, install and configure services, configure iptables rules and more. It's a great overview.
Install Kali on the Raspberry Pi 3. You could use a custom image, but if you do, you might have more troubleshooting to do. If you're unsure, use the stock image, which this solution was written for.
Implement WPA2 security on the AP.
Configure eth0 as DHCP, and wlan0 as static.
Configure the Raspberry Pi as a DHCP server for any wireless clients and assign a DHCP pool with 12-hour leases.
Have the SSH server start up at boot time so you can SSH to the Raspberry Pi once it's booted.
Forward all outbound traffic, including DNS, from wlan0 to eth0.
Allow inbound established (stateful) connections from eth0 to wlan0.
Hint: Although you have not learned about hostapd or dnsmasq, you will use them in this exercise.
Partial cheat: Although this article was not written for Kali (and will not work as written on Kali), it is the inspiration for this exercise, and is worth review. Thanks to Phil Martin for the inspiration.
Raspberry Pi 3. You can use an older model with a USB wifi but you're on your own when it comes to configuring wlan0.
Next, let's configure hostapd with a new configuration file. Note that an SSID and passphrase are configured for your access point.
This shows a successful run. Note that the errors pertaining to monitor mode are not relevant to us. For a RPi3 using the nexmon firmware, we would need (or an app) should nexutil -m2.
Using interface wlan0 with hwaddr b6:ae:d7:42:a1:70 and ssid "Kali-Pi3"
Note that your client may disconnect and reconnect because it didn't get an IP address. This is normal. You won't get an IP address until we configure dnsmasq. Have fun with this! It gives you an idea of how this process works, behind the scenes.
Press Ctrl-C to stop hostapd.
The forward isn't quite enough to give our wifi guests Internet access (through our eth0 interface). We need iptables to help us do this.
Whenever a new connection is encountered (-t nat), we want to alter the packets as they are about to go out (-A POSTROUTING) on our ethernet interface (-o eth0). The -j MASQUERADE target masks the private IP address of the client with the external IP address of the firewall/gateway (Kali Pi).
Next, we append (-A) a rule to the FORWARD chain (packets being routed through the Pi) which accepts (-j ACCEPT) packets from eth0 to wlan0 (-i eth0 -o wlan0) that belong to (ESTABLISHED) or are related to (RELATED) an existing connection.
Lastly, we will forward (and accept) all packets from wlan0 to eth0.
hostapd.service is not a native service, redirecting to systemd-sysv-install.
Finally, reboot and make sure the rules stick after a reboot. Once rebooted, you should be able to connect to the "Kali Pi" and surf!


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Portland Bulldogs Spring Sports Preview
By Michael Hoffer
Senior Donnie Tocci returns as the ace pitcher for a Portland baseball team hungry to win it all this spring.
File photos.
More photos below.
Coach: Mike Rutherford (17th year, 244-78 overall record, one state championship)
2018 record: 13-5 (Lost, 9-6, to Gorham in Class A South semifinals)
Top returning players: Will Barnard (Senior), Jake Loranger (Senior), Ben Sawyer (Senior), Ben Stasium (Senior), Donnie Tocci (Senior), Sonny Villani (Senior), Henry Westphal (Senior), Sam Knop (Junior), Brian Riley (Junior), Dante Tocci (Sophomore)
Pivotal games: April 27 FALMOUTH, April 30 TA, May 11 @ South Portland, May 18 CHEVERUS, May 25 SCARBOROUGH, May 28 @ Deering
Coach's comment: "We have a talented, senior-oriented group. The kids are confident. They've been together for four years. They just have to go out and do it. Replacing Cam King at catcher and Will Snyder at third base is key. It's a decent league, but not as strong as it was last year. I feel good that we'll score runs, play good defense and get good pitching. We have to be able to score runs off other teams' aces."
The Forecaster's forecast: Portland was right near the top of the league a year ago, thanks to its formula of good pitching, timely hitting and strong defense, but the defense abandoned the Bulldogs in their semifinal round playoff loss at Gorham. Portland returns most of their key players, but there are a few holes to fill. Regardless, this senior-laden squad is hungry to go out and win the program's first title in 21 years and if everything comes together, a championship celebration could be the end result.
On the mound, the Bulldogs will be paced by Donnie Tocci (a first-team SMAA all-star in 2018), whom Rutherford considers the best all-around player in the league, Villani (who will be the designated hitter when he's not pitching) and Riley. Replacing all-star catcher Cam King will be a challenge, but Portland will attempt to do so by using either Knop or Dante Tocci. The infield is experienced and will be a strength, as Portland emphasizes defense probably more so than any team. Sawyer (an honorable mention all-star last spring) plays first base, Westphal is at second, Barnard plays shortstop and either Dante Tocci or junior Cole Walter will step in to replace the graduated Will Snyder at third. Donnie Tocci plays rightfield when he doesn't pitch. Stasium (a first-teamer last year) is a rock in center and Loranger is very good in leftfield.
The Bulldogs have the pitching and defense to succeed. If the hitting can produce in the biggest games, this team will finish where it hopes to. Atop the heap. Nothing comes easily in Class A South, but Portland has been building for this season and won't settle for anything shy of a title.
Coach: Robbie Ferrante (fifth year, 40-31 overall record)
2018 record: 10-8 (Lost, 7-1, to eventual state state champion Scarborough in Class A South quarterfinals)
Top returning players: Grace Stacey (Senior), Laini Legere (Junior), Callie Watson (Junior)
Pivotal games: April 29 @ Noble, May 1 MASSABESIC, May 8 SOUTH PORTLAND, May 9 @ Scarborough, May 15 @ TA, May 17 BIDDEFORD
Coach's comment: "We have some turnover this year, but the kids are working hard. The kids coming back are excited. They know our winning ways and hope to pass it on. If we win games we should win early on, that will tell us how good we can be. I think we'll be in the middle of the pack."
The Forecaster's forecast: It's a new era for the Bulldogs, who graduated two pitchers, not just two-time Spring Female Athlete of the Year Jess Brown, but Sydney Gilbert as well. Portland does have some talent back and a few key newcomers should keep the Bulldogs in contention in Class A South.
Senior Jill Joyce, who comes over from the erstwhile Maine Girls' Academy, is expected to see a lot of time on the mound. She'll be a top hitter as well. Freshman Jordan King will also have an opportunity to hold the opposition at bay. Legere is back for her third season behind the plate. She wields a potent bat as well. Watson moves from first base to shortstop. Stacey at third base is another proven commodity. Both girls will help pace the offense also. Freshman Vanessa Connolly will play first base and could make an impact.
The program has lost a lot of talent to graduation over the past couple of seasons and while Portland isn't rated as high as teams like Scarborough and Thornton Academy, it should be able to contend with just about everybody. If the new pitchers can hold their own, the Bulldogs will post another winning record and could be a dangerous out when the postseason begins.
Coach: Mike DiFusco (seventh year, 33-44 overall record)
2018 record: 12-2 (Lost, 15-6, to Falmouth in Class A North Final)
Top returning players: Evan Bay (Senior), Cam Clifford (Senior), Nico Leavitt (Senior), Miki Silva (Junior), Ben Thompson (Junior), Max Cheever (Sophomore), Sam Gerber (Sophomore), Drew Leonard (Sophomore)
Pivotal games: May 10 @ York, May 24 WINDHAM
Coach's comment: "We graduated leadership and talent at every position from last year, but we've got a talented group returning, looking to step up for the program. Our defense and midfield are strong, so moving forward, we'll be counting on them to build around. On offense, our go-to guy is the open guy, so if we play unselfishly, we'll have a lot of success. It's a good group of kids with a lot of potential. Our goal by season's end is to be a confident and hungry team, having fun and ready for a playoff run."
The Forecaster's forecast: Portland awakened the echoes last spring, enjoying its best season since 2010, making a spirited run to the Class A North Final before finally meeting its match. Graduation took its toll on the program (all-star goalie Aaron Hoekstra and all-star attack Reilly O'Brien were among those departing), but the Bulldogs are out to prove they're consistent contenders and enough talent remains for that to happen.
Leavitt was a an honorable mention all-star in 2018 and he returns to lead the defense. Cheever (a longstick middie), Thompson, along with senior Koa Farnsworth and freshman Carlos Braceras, will be other key defensemen. Sophomore Johnny Sylvain replaces Hoekstra in goal. On offense, Bay, Clifford and Farnsworth will all get an opportunity to win possession in the faceoff circle. Bay, Clifford, Leonard and Silva are veteran midfielders. On attack, Gerber and senior Grant Jacobson, a transfer from Windham, are expected to lead the way.
Once again, Portland has a favorable schedule. The Bulldogs now expect to contend and won't be satisfied unless they post a winning record and make another deep playoff run. That's a likely final chapter for this squad.
Coach: Beth Broderick (third year, 14-13 overall record)
2018 record: 10-4 (Lost, 13-8, to Cheverus in Class A North semifinals)
Top returning players: Sydney Girsch (Senior), Chloe Kilbride (Senior), Elena Clifford (Junior), Annika More (Junior), Isabella More (Junior), Hazel Praught (Junior), Lam Zimet (Junior)
Pivotal games: April 22 WINDHAM, May 7 DEERING, May 23 @ Cheverus
Coach's comment: "We graduated eight seniors, including our all-star goalie, but we have a strong junior class. It's a great group of kids. The question is can we stay healthy and focused? The goal is to be mentally tougher than in the past. We put the pieces together the past couple years and hopefully we can take it further this year."
The Forecaster's forecast: Portland posted the second-best record in program history a year ago and could be even stronger this season.
The Bulldogs will be solid in the midfield behind Girsch, a captain, Praught, and the More twins, who were each named honorable mention league all-stars in 2018. Kilbride, another captain, who is heading to Plymouth State next year, leads the attack and figures to score plenty of goals. On defense, Clifford and Zimet will lock down the opposition in front of sophomore Samira Doiran, who replaces Abby Krieckhaus in goal.
Defending state champion Falmouth is the prohibitive favorite in Class A North, but Portland believes it could be the team best equipped to knock off the Yachtsmen by the time the season nears its end. The Bulldogs should continue their steady climb this spring and will again be one of the last teams standing in the region.
OUTDOOR TRACK
Boys' Coach: Dan Deniso (third year)
Girls' Coach: Bill LeRoy (fifth year)
(Boys) No score @ Class A state meet
(Girls) tie-19th @ Class A state meet
Top returning athletes:
(Boys) Joe Harrington (Senior), Will Brewster (Junior), Wyatt Dana (Junior), Sam Bibeau (Sophomore), Milo Bloom (Sophomore), Nate Fogg (Sophomore), Mwandja Kaluta (Sophomore), Jack Mahoney (Sophomore)
(Girls) Jaidyn Appel (Senior), Inez Braceras (Senior), Lucy Medd (Junior)
Coach Deniso's comment: "We want to increase our regional and state qualifiers and improve upon consistent daily preparation."
Coach LeRoy's comment: "We have good numbers in our freshman and sophomore classes. Hopefully they'll gain experience to turn in good performances. We're untested, but the girls are working hard. We'll see how they come along. Hopefully a few will make it to states."
The Forecaster's forecast: Both Portland outdoor teams are capable of showing great improvement in the weeks to come.
The boys' squad has enough athletes to cover most events. Brewster, Dana and Harrington are experienced distance runners. They're joined by freshmen Nate Brewster, Eli Parker, Jeremiah Senita and George Theall. Bibeau and Fogg are sprinters. They're joined by senior Keto Tchiputo and freshmen Philip Martin and Kevin Wang. Bloom, Kaluta and Mahoney compete in the sprints and hurdles, as will freshman Basillio Mangombo. On the field side, Bloom, Fogg, Kaluta, Mahoney and Mangombo all look to score in the jumps. Bibeau is a top thrower. If some of this talent develops as hoped, Portland will be strong in the regular season and could score some points at the state meet.
On the girls' side, Appel is the top returner. Portland's Winter Female Athlete of the Year, was runner-up in the high jump last spring and should be among the best jumpers in Class A again. Braceras and Medd are top distance threats. Freshman Anna Napiolo will look to make a mark in the sprints and the jumps. Several other youngsters have a chance to step in and make a name for themselves and help the program build for future success. Look for Appel to have a great year and for the Bulldogs to steadily improve as the season moves along.
Coach: James Siegel (sixth year)
2018 record: 13-2 (Lost, 5-0, to eventual state champion Falmouth in Class A South Final)
Top returning players: John Moran (Senior), Will Jorgenson (Junior)
Coach's comment: "We always look to improve, stay healthy and compete as well as we can. After losing so many players who were on the team for four years, we are rebuilding with a cadre of younger players and a few seniors vying for a couple doubles positions."
The Forecaster's forecast: Portland is coming off a terrific campaign, reaching the regional final for the first time since 1995, but the vast majority of that team has graduated, leaving some question marks as a new season looms. Don't write this group off, however.
Moran, a captain, is in the mix for one of the singles spots. He'll likely be joined by freshman Dominick Walls, who projects to be number one, and sophomore Brendan Mailloux, a transfer from Massachusetts. Jorgenson, the other captain, will be a key doubles player. Several other kids will have an opportunity to step in and make a mark.
Portland might not finish with the same record as last year, but the Bulldogs will remain very competitive in the league. An eighth successive playoff berth figures to follow and in the postseason, this is a group that no one will want to face.
Coach: Bonnie Moran (12th year)
2018 record: 9-7 (Lost, 4-1, to eventual state champion Falmouth in Class A South Final)
Top returning players: Karina Boothe (Senior), Kate Bourque (Senior), Lauren Paradise (Senior), Hannah Smith (Senior), Erin Chadbourne (Junior), Rose Watson (Junior)
Coach's comment: "This should be a good year for us. We only graduated number three singles and one of our doubles players, and we have the addition of Jenna (Lederer) who has the potential to be a very good player. Once again we have a large team, so we're working very hard to teach basic skills to beginners and improve the skills of the rest of the players who made a lot of progress last year."
The Forecaster's forecast: Portland made a stirring and unexpected run to the regional final last spring and even got a point off perennial champion Falmouth before bowing out. This time around, the Bulldogs figure to have an even better record, as well as a great chance to play into June again.
Paradise, a first-team all-SMAA selection and Portland's Spring Female Athlete of the Year a year ago, will be in the first singles position. Boothe returns at number two singles. Freshman Jenna Lederer shows great promise and will be in the third singles spot, the first freshman to play singles for the Bulldogs since Paradise three years ago. Bourque, Chadbourne, Smith and Watson all project to play doubles.
Falmouth remains the team that everyone is chasing, but Portland is hopeful it can end the Yachtsmen's long title reign. This squad will be fun to watch, will win a lot of matches and once again, should be at its best when the postseason arrives.
Sports Editor Michael Hoffer can be reached at [email protected]. Follow him on Twitter: @foresports.
Senior Ben Sawyer, an honorable mention all-star in 2018, is back at first base for Portland's baseball team.
Senior centerfielder Ben Stasium was an all-star in 2018 and will anchor the outfield, as well as make things happen at the plate and on the basepaths.
Senior Sonny Villani is another top hurler for Portland.
Senior Henry Westphal is a solid second baseman for the Bulldogs.
Junior Laini Legere is back for her third season as the catcher for Portland's softball team. She'll also wield a potent bat.
Junior Callie Watson will play shortstop this spring for the Bulldogs and will be one of the team's top hitters.
Senior midfielder Cam Clifford returns looking to help Portland's boys' lacrosse team enjoy another memorable season.
Sophomore long-stick middie Max Cheever will make life difficult for the opposition.
Junior Miki Silva will be a matchup problem in the midfield.
Junior Elena Clifford is a top defender for Portland's talented girls' lacrosse team.
Junior Isabella More will be in the middle of the scoring frenzy for the Bulldogs.
Junior Hazel Praught is another key returner for the Bulldogs.
Senior Lauren Paradise will play first singles for a Portland girls' tennis team which reached the regional final in 2018.


text:
Bankrupt tennis legend Boris Becker puts his trophies and souvenirs up for auction
By Ishan Ghosh
Boris Becker Reuters
Six-time Grand Slam champion Boris Becker's trophies and personal souvenirs are put on auction from Monday, June 24, by a British firm named Wyles Hardy to partially clear of the debts of the bankrupt tennis legend. The former tennis star who was popularly known as 'Boom Boom' Becker for his devastating serves is auctioning 82 items which include medals, cups, photographs and watches. The sale will end on July 11, according to the firm's website.
Becker, who won his first Grand Slam at the age of 17, was declared bankrupt in 2017. In 2018, he was supposed to auction his trophies and souvenirs but he claimed to have a diplomatic status which stopped the sale.
According to the 51-year-old former world number one, he had been assigned by the President of the Central African Republic as a specialist for sports, culture and human welfare. But the Central African Ministry of Foreign Affairs in response said that the passport owned by Becker was fake. So, it did not work out and finally Becker auctioned his trophies on the market.
According to Mark Ford, one of the three trustees of Becker's bankruptcy estate, "The first attempt "attracted very substantial bids." But the sales will not be enough to cover his debts which amounts to millions of pounds.
The legendary tennis player who won 49 career titles is also working as a commentator to try and pay off his debts. The items that are to be auctioned include a replica of Challenger Cup that was awarded to the German tennis star after one of his wins at Wimbledon. Another trophy that features in the list is the replica of the Renshaw Cup, which he won after becoming the youngest player ever to win a Grand Slam. His Wimbledon finalist medal from the 1990's edition of the tournament and a replica of the US Open silver cup from 1989 are also up for bidding.
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The La Roja have been performing pretty well at the starting of the...


It was great to spend time with Principal Brad Boudreau, Assistant Principal Georgette Knecht, and Assistant Principal Kara Lee at Pine Middle School. Pine MS has established core beliefs that drive their work. They strongly focus on high expectations for all students and staff members. They are data driven and focused on Tier I Instruction.
Principal Josh Reddig welcomed me to the Academy of Arts, Careers, & Technology for a visit with him, his staff, and their students. Principal Reddig is proud and enthusiastic about the robust activities at AACT and their student engagement. He shared details about many of the events the students are preparing for and the important planning and coordination by their staff.
Principal Joel Peixoto and the Dilworth STEM Academy office staff welcomed me during my recent visit to their school. Principal Peixoto and his staff have created a warm, welcoming environment that includes a computer station and coffee for visiting families and guests. During my visit, I learned about their focus on family involvement and the high level of family engagement they enjoy at Dilworth.
I spent time at Towles Elementary School with Principal Rhonda Van Deusen and the Towles Superstars. This neighborhood school has a welcoming, friendly atmosphere. It was great to hear about the activities at Towles and visit with students and staff members.
Principal Bruce Meissner spent time talking to me about how special Clayton Pre-AP Academy is, along with his admiration for the staff and students. He told me about the structure and history of Clayton over the last decade and how they focus on student achievement and well-being. I enjoyed hearing about the culture and climate at Clayton along with the work they are doing to achieve academic success and academic growth for all students.


Last year, a French booksellers' union took Amazon to court claiming its free shipping violated the 1981 Lang Law, which forbids booksellers from offering discounts of more than 5 percent off the list price. Apparently, Amazon was exceeding that discount when the free shipping was factored in, thus the Tribunal de Grande Instance in Versailles awarded the union €100,000 and ordered Amazon to start charging for shipping within ten days.
But the giant online retailer said that it would pay €1,000 a day in fines, rather than comply with the court ruling, while it appeals to keep its shipping free in France. The French legal system will only allow Amazon to pay the €1,000 fine for 30 days, at which point the court can extend the fine, lower it or raise it. But considering the law has twice come before the European Court of Justice and was upheld in both instances, Amazon's appeal may stand little chance of success.


The Edmonton Fencing Club will be hosting two open houses this year - September 5th, 12-4pm and September 11th, 4-8pm. The open houses are a great opportunity to check out the facility, try fencing and obtain information on fencing classes and prices for the upcoming fall term. All friends and families are welcomed!


CEDAR RAPIDS, Iowa – The Evangel men's basketball team was unable to keep pace with a hot-shooting Mount Mercy squad Saturday falling 106-81 in Heart of America Athletic Conference play.
The Crusaders managed to hold the Mustangs, the nation's top three-point shooting team, to just nine three-pointers in the loss. It was the third lowest total of the season for Mount Mercy, however, the Mustangs came out hot shooting 56 percent from the field in the first half while Evangel shot 36 percent.
Evangel also managed just four three-pointers in the game as Clark Brewington went 3-of-9 from behind the arc and led the team with 20 points. Chris Stocks also finished in double figures adding 17 points.
Cade Coffman was shy of a double-double tallying eight points and 13 rebounds. Jacob Smith added 10 points for the Crusaders off the bench as well.
Mount Mercy was led by Dennis McKinney who scored a game-high 25 points.
Evangel continues road action Wednesday at No. 3 Benedictine who sits atop the league standings at 18-0. Tipoff from Atchison, Kansas is scheduled for 7:30 p.m.