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The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin–antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle.
Antimicrobial resistance is a major threat to global health1. Biofilms are resistant communities of microorganisms attached to surfaces and encapsulated in a matrix2,3. Biofilms are involved in 80% of human bacterial infections4 and confer antibiotic resistance5.
Oxygen consumption in the biofilm generates oxygen gradients6,7 and may be linked to biofilm dispersal8. Selective cell death, for example, in anoxic regions, creates channels that facilitate nutrient-waste product exchange9,10,11 and biofilm dispersion12. Dispersed cells are more susceptible to antibiotics than those in biofilms, although they are also distinct from planktonic cells in terms of gene expression and pathogenicity13.
Classical toxin–antitoxin (TA) systems are based on silencing of a stable toxin by a labile antitoxin that, when inactivated, releases the toxin, resulting in a reduction in metabolism. TA systems modulate the generation of persister cells14, phage inhibition15 and biofilm regulation16.
The Hha/TomB TA system is part of the first group of TA systems identified in biofilms17. The overexpression of Hha (haemolysin expression modulating protein) causes cell lysis and reduction of biofilm formation as well as increases biofilm dispersal18. An engineered variant of Hha caused nearly complete biofilm dispersal via cell lysis19,20, and deletion of hha led to no dispersal of Escherichia coli BW25113 (ref. 20) corroborating the role of Hha in biofilm dispersal. TomB (toxin overexpression modulator in biofilms, previously known as YbaJ) inactivates Hha toxicity18 although the mechanism has not been elucidated.
Here we report that the TomB antitoxin activity is oxygen-dependent and that its Yersinia orthologue, YmoB can replace TomB as the Hha antitoxin. A single cysteine variant [C117S]YmoB is similarly active in vivo and promotes the oxidation of the single conserved cysteine of Hha to -SOxH (sulfenic, sulfinic and sulfonic acids) by air in the absence of any external source of reactive oxygen species. Oxidation of Hha or the introduction of a negative charge at the position of the cysteine residue, mimicking the presence of -SOx−, causes its destabilization and results in reduced toxicity.
The three-dimensional (3D) structure and also the Hha transient interaction site of [C117S]YmoB were determined by nuclear magnetic resonance (NMR). Relevant structural features include a buried cysteine residue located close to the Hha-binding site and connected to the opposite side of the four-helix bundle by a narrow channel with a width comparable to that of an oxygen molecule. The built-in oxygen sensor in this TA system may provide a mechanism for environmental regulation of cell activity.
Taken together, these observations suggest that Hha/TomB represent a TA system based on the inactivation of the toxin (Hha) by oxidation with molecular oxygen mediated by the antitoxin (TomB).
We tested the hypothesis that the Hha-TomB pair was an oxygen-dependent TA system by measuring E. coli growth at increasing agitation rates (Fig. 1). Overexpression of Hha has a toxic effect in minimal medium, resulting in a decrease of the growth rate and the cell density in the stationary phase. Growth in rich medium showed an even higher toxicity18. Simultaneous overexpression of Hha and TomB in minimal medium results in biphasic growth gradually evolving from the slow growth rate observed when Hha is overexpressed in the absence of TomB to the faster growth observed in the controls not expressing Hha. A significant antitoxin effect of TomB was observed after ∼4 h, and after 6 h, cultures overexpressing both Hha and TomB reached the same cell densities as the controls.
Figure 1: TomB antitoxin activity depends on the agitation rate.
(a) Growth curves of E. coli K-12 MG1655 Δhha cells harbouring pCA24N-hha and pBAD30-tomB at (from top to bottom) 100, 150, 200 and 250 r.p.m. agitation rates. Control cultures with no expression of Hha or TomB (squares) were compared with cultures expressing only Hha (circles) and co-expressing both Hha and TomB (diamonds). Growth curves were measured in triplicate at 37 °C in tryptone minimal medium (TMM; 10 g l−1 tryptone and 2.5 g l−1 NaCl). Error bars represent the sample s.d. (b) Growth curves and simultaneous measurements of the oxygen saturation during culture growth at 150 r.p.m. (top) and 250 r.p.m. (bottom) agitation rates.
Experiments at the two extreme agitation rates were repeated while simultaneously measuring oxygen saturation, using an oxygen-sensitive polarographic electrode (Fig. 1b). Oxygen levels reflect the balance between air uptake, enhanced by fast agitation, and oxygen consumption by the metabolic activity of the bacteria. At an agitation rate of 100 r.p.m., the oxygen saturation levels of the control and Hha+TomB expressing cultures reached a minimum of 1% while at an agitation rate of 250 r.p.m., the oxygen levels were always higher than 30%. Oxygen levels increased when the cultures approached the stationary phase. The longer lag time for the antitoxin activity at slow agitation rates is consistent with oxygen being required for TomB activity as an antitoxin.
At the agitation rate of 250 r.p.m., the oxygen levels of the cultures overexpressing only Hha were always higher than the controls, reflecting a lower oxygen consumption in the presence of the toxin. At 100 r.p.m., cultures expressing Hha+TomB or only Hha reached similar oxygen levels (10% saturation) in late stationary phase in spite of the fact that the cell density, as measured by the turbidity at 600 nm, was lower in the cultures expressing only the toxin. However, in these cultures, the number of colony forming units (CFU) was lower than in those expressing also the antitoxin (Supplementary Table 1). These observations suggest that 10% oxygen saturation is enough to enable TomB antitoxin activity. Interestingly, the observation that the number of CFU is higher in the samples overexpressing the two proteins (Hha+TomB) than in those only expressing Hha rules out the possibility that the observed toxicity of Hha is only related to the metabolic stress associated with protein overexpression and confirms that the toxicity of Hha can be compensated by TomB.
These observations suggest an oxygen-dependent TA mechanism that we investigated in detail by combining chemical biology and structural studies.
A non-redundant collection of 250 TomB homologues was obtained by BLAST search using as a query the TomB sequence from E. coli K-12 strain (D64776). The sequences (Supplementary Fig. 1), representative of the YbaJ-superfamily, contain from one to five cysteines. Cysteine 18 (in 244 sequences) and a second cysteine located towards the C-terminus (235/250 sequences) are most conserved. Attempts to obtain suitable constructs to carry out structural studies of TomB failed, so we focused on its Yersinia’s homologue YmoB, which has 58.1% pairwise identity, 73.6% pairwise similarity and contains the two most conserved cysteines (C18 and C117, Fig. 2a). Its single cysteine variant [C117S]YmoB proved suitable for structural determination by NMR, as shown below.
Figure 2: Yersinia’s YmoB protects E. coli from toxic effect of Hha.
All experiments were carried out using E. coli K-12 MG1655 Δhha cells harbouring pCA24N-hha and pBAD30-tomB, pBAD30-ymoB or pBAD30-ymoB(C117S), at 37 °C and in TMM. (a) Sequence alignment of TomB/YmoB proteins. Asterisks highlight the position of conserved cysteine residues at positions 18 and 117. (b) Growth curves of control cultures with no expression of a TA pair of proteins (squares) were compared with cultures overexpressing: Hha (circles), Hha and TomB (diamonds), Hha and YmoB (triangles up) or Hha and [C117S]YmoB (triangles down). Experiments were performed in duplicate, and the error bars are the sample standard deviation. (c) Microtitre plate biofilm assay. Total biofilm formation was measured at OD540nm and was standardized as OD540nm/OD620nm. Experiments were performed in duplicate, and the error bars are the sample standard deviation. (d) Representative IMARIS (BITplane, Zurich, Switzerland) images of flow cell biofilm with Hha overexpression (up) and Hha and YmoB co-expression (down). Dead cells (left) are shown in red and live cells (centre) in green, while superimpositions of dead and alive cells are shown on the right. OD, optical density.
We first explored if YmoB and [C117S]YmoB could functionally replace TomB in preventing Hha-associated toxicity in E. coli in planktonic cells and in biofilms. Planktonic cell cultures overexpressing Hha and either of the two YmoB variants reached the same cell density of the controls not expressing Hha or overexpressing both Hha and TomB (Fig. 2b), although the antitoxin activity of the YmoB variants showed a longer lag time than TomB. The C117S substitution had no significant effect on YmoB antitoxin activity in planktonic cells.
Overexpression of Hha causes a fourfold decrease of standard biofilm formation (Fig. 2c). The co-expression of TomB or YmoB totally restored biofilm formation, although the C117S variant was slightly less efficient. Regarding biofilm morphology, Hha overexpression leads to amorphous biofilms with a higher percentage of dead cells (Live/Dead cells ratio of 5.6±1.4, quantitative analysis by COMSTAT21 software). Instead, when YmoB was co-expressed, the Live/Dead cell ratio significantly increased to 35.4±8.5 and the biofilm was composed by microcolonies uniformly spread on the entire surface (Fig. 2d). Thus, YmoB or [C117S]YmoB can functionally replace TomB as the Hha antitoxin in E. coli.
Hha has a single cysteine residue (C18) highly conserved in the Hha-family of proteins. We hypothesized that the oxygen-dependent antitoxin activity of TomB and [C117S]YmoB towards Hha could be related to the oxidation of this conserved cysteine residue.
In addition to their capacity to form disulfide-bonds, cysteine thiol groups can be oxidized to sulfenic (-SOH), sulfinic (-SO2H) and sulfonic (-SO3H) acids22. Sulfenic acid formation is reversible but sulfinic and sulfonic acid are irreversibly formed and result in the introduction of a negative charge. Hha has an unusual high proportion (33%) of charged residues and its structure is highly sensitive to electrostatic effects23. The additional charge introduced by cysteine oxidation could destabilize Hha and prevent its inherent toxicity.
To investigate the effect of YmoB on the oxidation of C18 of Hha in vitro, we used mass spectrometry. Identical samples of Hha were incubated in the presence and in the absence of [C117S]YmoB for 30 min. Hha was isolated by size exclusion chromatography (SEC), digested with trypsin and analysed by mass spectrometry to identify cysteine-containing peptides in their reduced and oxidized (-SOxH) forms. Figure 3a shows a decrease in the reduced form and an increase in the oxidized forms of Hha in the presence of [C117S]YmoB. The differences with the untreated Hha samples are statistically significant according to a Student t-test with P=0.03 (for sulfonic acid) and P=0.02 (reduced and other oxidized forms). Variants of YmoB in which F111 had been mutated showed no effect (Fig. 3b). The choice of the phenylalanine mutants was based on the structural studies described below. Interestingly, even in the absence of YmoB, Hha contains a significant proportion of peptides with oxidized forms of cysteine suggesting that the effect of YmoB is to enhance its spontaneous oxidation. The Hha homologue in Yersinia, the protein YmoA, showed similar oxidation effects (Supplementary Fig. 2).
Figure 3: [C117S]YmoB enhances the oxidation of Hha.
The bars represent the percentage of peptide spectrum matches (PSM) to the various oxidation states relative to the total PSM of cysteine-containing peptides in the mass spectra of trypsin-treated samples of Hha. Parallel experiments were performed by incubating Hha for 30 min without any added YmoB variant, or in the presence of (a) [C117S]YmoB, or (b) [F111L,C117S]YmoB or [F111Y,C117S]YmoB. The experiments were run in triplicate and each sample was analysed in duplicate. The error bars are the standard deviations. Asterisks mark statistically significant deviations according to a Student t-test with P=0.02. [C117S]YmoB enhanced oxidation of YmoA was measured under different conditions and is shown in Supplementary Fig. 2.
We mimicked the effect of adding a negative charge at the cysteine position by mutating it to glutamic acid, and we measured the growth of E. coli Δhha expressing the [C18E]Hha variant. As controls, we used wild-type Hha and a previously studied C18I variant24. While [C18I]Hha retains its capacity to act as a toxin, the presence of a negative charge at this position substantially reduces the toxicity of the C18E variant (Fig. 4a).
Figure 4: A negative charge in position 18 of Hha destabilizes the toxin.
The negative charge introduced by cysteine oxidation was mimicked by the C18E mutation. (a) Growth curves of E. coli BW25113 Δhha cells harbouring: pCA24N (squares), pCA24N-hha (circles), pCA24N-hha(C18E) (triangles down) and pCA24N-hha(C18I) (triangles up). Each data point represents the mean of three independent cultures, and error bars denote s.e.m. (b) E. coli BW25113 Δhha cell lysates after 4 h growth harbouring: pCA24N (empty), pCA24N-hha (Hha), pCA24N-hha(C18E) (Hha C18E) and pCA24N-hha(C18I) (Hha C18I). Arrow denotes Hha and its mutated variants. For each strain, 25 μg of total protein was loaded on 18% SDS-PAGE. Experiments performed at 37 °C in LB medium. (c) 1H–15N HSQC NMR spectra of Hha (red) and [C18E]Hha (black). The reduced spectral dispersion in the spectra of the mutated protein indicates a loss of tertiary structure. (d) Changes in NMR peak intensity of native Hha treated with hydrogen peroxide. The intensity changes of peaks in helix 1 are indicated in red, while peaks in other regions are shown in blue. (e) Hha structure with helix 1 and cysteine 18 highlighted. SDS–PAGE, SDS–polyacrylamide gel electroporesis.
The expression levels of wild type, C18I and C18E variants of Hha are comparable after 4 h of induction at 37 °C (Fig. 4b). However, when [C18E] Hha was overexpressed and purified, this variant showed a complex NMR spectrum with low dispersion and non-uniform peak intensities typical of a denatured protein (Fig. 4c), while the C18I mutant had been previously shown to be well folded24. Thus, the introduction of a negative charge at position 18 destabilizes the structure of Hha. A similar effect had been previously observed in Hha, when changing the electrostatic balance by the removal of a negative charge via the substitution E53Q, which resulted in complete protein denaturation25.
Hha was also treated with hydrogen peroxide in vitro to induce its oxidation. The NMR spectra of 15N-labelled Hha showed a clear decrease in intensity over time after the addition of H2O2 (Fig. 4d). The most-affected region is centred in a helical segment next to the cysteine residue (Fig. 4e). Helix 1 contains a methionine residue that can also be oxidized. The loss of intensity indicates a structural change from the native form. A dynamic molten-globule type structure or soluble aggregates are two of the obvious possibilities. Dynamic light-scattering experiments showed no significant variation after treatment with H2O2 suggesting the absence of aggregation (Supplementary Fig. 3).
Taken together, the in vivo and in vitro results confirm that YmoB variants or TomB act as antitoxins by enhancing the inactivation of Hha by oxidation.
To gain molecular level insight into the role of [C117S]YmoB in the oxidation of Hha, we decided to determine its 3D structure. No structure of any member of the YbaJ-superfamily is yet known.
Although the [C117S]YmoB variant provided NMR spectra of enough quality to complete the backbone assignment (Supplementary Fig. 4), slow time evolution hindered the assignment of the side chains associated NOE (Nuclear Overhauser Effect) cross-peaks required for structure determination. Therefore, the suite of NMR experiments needed to determine the 3D structure of [C117S]YmoB were carried out in interleaved mode using non-uniform sampling and targeted acquisition26. The 3D structure was determined using 1349 NOE/distance, 198 dihedral angle and 36 hydrogen bond restraints with UNIO’10/CYANA 2.1 software27,28,29 and final refinement in the presence of explicit water with CNS 1.2.1 software30,31. Refinement statistics are summarized in Table 1. The NMR assignment and the structural models have been deposited in the RCSB PDB with accession numbers RCSB104115 and 2MN2. Details of the process, experiments and NMR calculation are presented in the Supplementary Tables 2 and 3.
Table 1: NMR constrains and refinement statistics.
Figure 5a shows the superposition of the 20 lowest energy models, which have a root mean square deviation considering all backbone heavy atoms of 1.89±0.45 Å, and 0.87±0.14 Å if only the residues in the α-helices are compared. [C117S]YmoB forms a compact antiparallel four-helix bundle connected by flexible loops. A one-turn helix H5, containing the C117 in the wild-type form, is located at the C-terminus.
Figure 5: NMR structure of [C117S]YmoB as a model for TomB structure.
(a) Superimposition of 20 lowest energy models. Helical regions (H1: 7–30; H2: 43–65; H3: 70–87; H4: 94–111; and H5: 116–119) are shown in white and non-structured regions in blue. C18 is coloured yellow. The same colour is used for H1 in the topological representations. Helices H3 (negatively charged) and H4 (positively charged) are represented in red and blue, respectively. (b) Surface representation of the residues broadened in the presence of paramagnetically labelled Hha. Those located in helices H2 and H3 are shown in red. Additional affected residues, (mainly in loops) are marked in orange (see Supplementary Fig. 4). The structures in a,b are rotated 180°. Most of the residues contacting Hha are located in one side of the structure. (c) Expanded view of the pocket and channel defined by conserved residues F108 and F111 (purple), K15 (blue), E19 (red) and S117 (C117 in wild-type YmoB, yellow) connecting the surface with the location of the sulfur atom of C18. M120, also susceptible to oxidation and close to C117, is shown in orange. The entry of the channel is located in the opposite face from the Hha-binding site. S117 is occupied by cysteine in the wild-type form of YmoB. (d) Logo representation of the sequence variability within the YbaJ-superfamily. Secondary structure of [C117S]YmoB is represented. An asterisk highlights residues forming the pocket surrounding C18. Some of the sequences terminate after position 116. The proportion of sequences that extended to a given position is shown and colour-coded in circles at the C-terminal end of the logo representation.
A singular feature of the structure is the location of the isolated cysteine residue (C18) in helix H1, which is completely buried in the protein hydrophobic core. Consistently, C18 is completely inaccessible to iodoacetic acid.
Typical TA pairs form stable complexes masking the toxin. However, addition of Hha or YmoA did not significantly affect the NMR spectra of 15N-labelled [C117S]YmoB. The NMR spectra of 15N-labelled Hha or YmoA were also not significantly affected by [C117S]YmoB. Thus, in this case, the toxin (Hha) and antitoxin (YmoB) components do not form a stable complex. However, they do interact transiently, which is enough to cause the chemical inactivation of the toxin.
This fact could be clearly demonstrated using paramagnetic relaxation enhancement NMR experiments. When a paramagnetic centre approaches, even transiently, to specific nuclei, NMR signals are strongly broadened due to fast relaxation and the corresponding decrease in signal intensity is easily measured32.
The native single cysteine residue of Hha was reacted with 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) that incorporates a nitroxide spin label. We compared the intensities of the signals from individual residues of 15N-labelled [C117S]YmoB in the presence and in the absence of paramagnetic Hha.
[C117S]YmoB residues perturbed by spin-labelled Hha are clustered: one of the most perturbed residues is E53 located in helix 2. Additional affected residues are located in adjacent turns of the same helix (N49, A56 and S57) and in three consecutive turns of helix 3 (E77, E78, D81 and Y84) (Fig. 5b and Supplementary Fig. 5). Of these, N49, E53, A56 and D81 are absolutely conserved in the YbaJ-superfamily. In addition, position 77 is always occupied by a negatively charged residue and position 84 by tyrosine or phenylalanine (Fig. 5d). The transient interaction of spin-labelled YmoA with [C117S]YmoB was similarly mapped and affected the same regions (Supplementary Fig. 5). The conserved E53 residue was mutated to lysine in TomB. The gene expressing [E53K]TomB or wildtype TomB were incorporated in the pCA24N-tomB-hha plasmid that also encodes the hha gene. The plasmids were incorporated into a Δhha ΔtomB host and the growth curves were compared. The strains with [E53K]TomB showed significantly higher growth confirming that the E53 residue is functionally important also in TomB (Supplementary Fig. 6). The better performance of the E53K variant suggests an improved antitoxin activity against Hha, although the possibility that TomB has some residual toxicity that is reduced in the E53K mutant cannot be ruled out.
A buried site in the interior of the four-helix bundle where the conserved C18 residue of YmoB is located is a candidate to be the protein ‘active site’ responsible for the enhanced reaction of Hha with molecular oxygen. The YmoB C18 residue is located close to the Hha binding site. However, its buried location raises the question of how oxygen can reach the cysteine site.
Analysis of the structure of [C117S]YmoB using the DoGSiteScorer software33 identified a shallow pocket on the surface of [C117S]YmoB formed by K15, E19, F108, F111, S112, G113, I116, S117 and M120 (Fig. 5c). In some of the NMR structures K15, E19, F108 and F111 side chains define a narrow channel that connects the protein surface with the location of C18. The width of this channel is comparable to that of an oxygen molecule, and we hypothesized that it could facilitate its access to the interior of the four-helix bundle. To test this hypothesis, we mutated F111 of [C117S]YmoB to leucine and to tyrosine. The NMR spectra of the two mutants are typical of a well-folded protein and, in the case of the F111L mutant, chemical shifts are very similar to the wild type suggesting a very similar backbone structure (Supplementary Fig. 7). However, these two YmoB variants lost their capacity to enhance Hha oxidation in vitro (Fig. 3b), in agreement with the suggested role of phenylalanine side chains in channel formation and YmoB-enhanced oxidation of Hha.
YmoB or [C117S]YmoB at 200 μM concentration were incubated in the presence of air, for 1 day at room temperature with 1:10 dimedone, a specific reagent for sulfenic acid detection yielding a stable adduct with a characteristic mass increment, in the presence and in the absence of 2 mM dithiothreitol (DTT). Disulfide-bonded oligomers were separated by SEC and monomeric samples were then digested with GluC endopeptidase. Samples were analysed by liquid chromatography coupled to tandem mass spectrometry to identify peptides containing oxidized cysteine residues. All possible SOxH oxidized forms were detected (Supplementary Fig. 8). In addition, the observation of a dimedone adduct of C117 in wild-type YmoB confirmed the formation of sulfenic acid. Although sulfenic acid was also detected by the mass increment of the corresponding peptides, no adduct with C18 was observed.
In order to test whether YmoB cysteine residues are essential for the antitoxin activity, we compared E. coli Δhha growth for cells overexpressing Hha and wildtype YmoB, [C117S]YmoB, [C18S,C117S]YmoB or no antitoxin (Supplementary Fig. 9). Strains co-expressing Hha and the cysteine-free YmoB variant showed a longer lag time than those co-expressing Hha and YmoB. Even so, the final cell density at the stationary phase reached the same level as in the controls not expressing Hha. Thus, the presence of cysteines is not essential for the antitoxin activity of YmoB, although they contribute to make it more efficient.
The four-helix bundle is highly conserved in a set of 250 TomB homologues while the C-terminal region, following helix H4, shows the largest variability (Fig. 5d and Supplementary Fig. 1).
One of the most conserved regions includes cysteine 18 and its environment: position 15 is exclusively occupied by lysine or arginine, position 19 is preferentially occupied by glutamic and aspartic acid and position 17 by leucine, placing C18 in a peculiar hydrophobic but polarized environment. The hydrophobic environment is completed by aromatic residues in position 108 (phenylalanine appears in 225/250 sequences) and in position 111 (phenylalanine in 240/250 sequences) forming an access channel to C18 in [C117S]YmoB (Supplementary Fig. 10).
The residues transiently interacting with Hha and their nearest neighbours are also highly conserved: E53, N49, A56, E77, E78, D81, D82 and F84.
Thus, the TA mechanism demonstrated in vivo for Hha/TomB and that is operative also for the artificial Hha/YmoB pair is likely to be a general feature in the entire YbaJ-superfamily. However, in spite of the large similarities, there are distinct subfamilies that differ, for example in the number of cysteine residues. In this respect, TomB and YmoB are not the closest members of the family but, nevertheless, YmoB could functionally replace TomB as an antitoxin. We also demonstrated similar oxidation processes in YmoA, suggesting that a similar TA system may also exist in Yersinia sp.
A homology model of TomB was built from the structure of [C117S]YmoB using Modeller34. TomB has four cysteines, in positions 18, 25, 110 and 124. C110 is located at the end of helix H4, not far from the location of cysteine 117 in YmoB, and C124, located at the C-terminus of TomB that extends beyond the end of YmoB.
TomB cysteines 18 and 25 are located in helix H1 and are both buried in the interior of the four-helix bundle. Interestingly C18 and C25 are located symmetrically, but in opposite directions, with respect to the Hha interaction site. The residues flanking C18 (Leu-18Cys-Glu) and C25 (Asp-25Cys-Leu) form a symmetrical pattern. The residues surrounding C18 in the 3D structure of [C117S]YmoB and in the homology model of TomB are identical.
The conservation of the buried cysteine residues, their location close to the Hha interaction site, and the fact that C18 in YmoB experiences similar oxidation processes as those observed in Hha suggest that this residue may be involved in the oxidation enhancement mechanism. However, the fact that a variant of YmoB without any cysteine retained some capacity to act as an antitoxin indicates that the cysteines of YmoB are not essential. On the other hand, mutations affecting the channel leading to the YmoB C18 site prevented the oxidation of Hha. Thus, the environment around YmoB C18, rather that the cysteine residue itself is probably sufficient for YmoB-enhanced oxidation, although the cysteine residue may play a role, for example through its transient and reversible oxidation to sulfenic acid. TomB seems to have this site duplicated while the Hha-binding site is not. These structural variations among the YbaJ superfamily probably reflect adaptions to the required levels of antitoxin activity or their oxygen dependency.
The cysteine residues of Hha and YmoA are partially oxidized spontaneously by air in vitro even in the absence of TomB or YmoB giving a mixture of sulfenic, sulfinic and sulfonic acid. However, the oxidation is enhanced in the presence of [C117S]YmoB. This cysteine residue is conserved in nearly all members of the Hha family but its function was unknown. It is located in a loop between to helical regions, only partially solvent exposed, and located in the interface between regions with opposite charge35,36. The unusual sensitivity to oxidation may be related to this particular environment. Interestingly, C18 in YmoB is also located in a buried region but surrounded by charged residues.
Reactions of free molecular oxygen have a high-activation barrier associated with the symmetry of its electron spin triplet ground state37. The presence of polar residues, electric dipoles or interactions breaking the local symmetry enhances the capacity of molecular oxygen to oxidize cysteine to SOxH species22,38.
Hha oxidation leads to a loss of structure thus explaining its inactivation by TomB in the presence of oxygen. The Hha structure was also destabilized by a mutation introducing a negative charge at the position of C18. The sensitivity of Hha to charge modifications had been previously observed and is consistent with its unusual high density of charged residues23,25.
Overall, these results strongly suggest that the TA system formed by proteins from the Hha-YbaJ superfamily acts through a mechanism that does not fall in any of the currently accepted TA classes. While known TA systems are based on a stable toxin that is neutralized by forming a complex with a labile antitoxin, the Hha-YbaJ system is based on the chemical inactivation of the toxin caused by oxidation, enhanced in the presence of the antitoxin. This process does not require the formation of a stable TA complex but takes place through transient interactions.
The described mechanism links the TA system to the concentration of oxygen, making proteins from the YbaJ-superfamily effective antitoxins only for highly oxygenated cells. Strong oxygen concentration gradients in biofilms could cause the antitoxin to be less efficient in cells located in anoxic regions, resulting in selective death of these cells and the creation of cavities in the biofilm matrix allowing the escape of planktonic cells, or detachment of entire microcolonies facilitating biofilm dispersion.
The structure of [C117S]YmoB provides hints on the chemical origin of this activity and suggests that the members of the YbaJ-superfamily could be targets for antibacterial treatments to inhibit the formation of biofilms, one of the strongest contributors to the pressing antibiotic resistance problem.
Growth curves. One colony of E. coli K-12 MG1655 Δhha cells harbouring pCA24N-hha (under the control of T5lac promoter, activated by 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside), selected with 100 μg ml−1 of ampicillin) and pBAD30-tomB or pBAD30-ymoB or pBAD30-ymoB(C117S) or pBAD30-ymoB(C18S)(C117S) (under the control of araC promoter, activated by 0.1% arabinose, selected with 50 μg ml−1 of chloramphenicol) was pre‐cultured overnight at 37 °C and 250 r.p.m. in 30 ml of tryptone minimal medium (TMM: 10 g l−1 tryptone and 2.5 g l−1 NaCl).
Growth curves were started by adding 4 ml of preculture to 200 ml of TMM (with corresponding antibiotics and chemical inducers) and the experiments were carried out with the incubators and the room maintained at 37 °C.
Oxygen saturation measurements. Dissolved oxygen was measured as the percentage of saturation using a polarographic electrode (VWR DO210) using the built-in temperature and ionic-strength compensation. The electrode was cleaned with 14% ammonium hydroxide for 2 min and MilliQ water for 1 min to prevent cross-contamination.
Colony forming unit measurements. The number of CFUs was obtained by platting 10 μl of a 108 dilution of samples taken at selected optical densities.
Microtiter biofilm assays. Single colonies of E. coli K-12 MG1655 Δhha cells harbouring pCA24N-hha and pBAD30-tomB or pBAD30-ymoB or pBAD30-ymoB(C117S) were inoculated into 25 ml TMM medium (with the corresponding antibiotic) and incubated overnight at 37 °C and 250 r.p.m. shaking speed. Two independent cultures were prepared for each strain, diluted with fresh TMM (with the corresponding antibiotic) to obtain an OD600 below 0.05, and 300 μl of culture were added into separate wells of a 96 well plate. Blanks were prepared with pure TMM medium. The plate was incubated at 37 °C without shaking, for 15 h. To record the total growth, we measured OD620. Supernatant was discarded and the plate was washed by dipping three times in MilliQ water, 300 μl of 0.1% crystal violet were added in each well and the plate was incubated for 20 min at room temperature. Staining solution was discarded and the plate was washed by dipping three times in MilliQ water. An amount of 300 μl of 95% ethanol was added to each well and was incubated for 5 min. We measured the total biofilm reading OD540 after mixing for 50 s. Standard biofilm formation was calculated as OD540/(OD620− OD620). OD measurements were done using Infinite M1000 PRO plate reader.
Flow cell biofilm assays. Strain E. coli K-12 MG1655 Δhha pCA24N-hha pBAD30-ymoB was streaked on LB agar plates with the corresponding antibiotics. One‐day old single colonies were inoculated in 25 ml of TMM overnight at 37 °C at 250 r.p.m. Overnight cultures were diluted to reach OD600 below 0.05 in 150 ml TMM with the corresponding antibiotics. Diluted cultures under stirring at 37 °C were pumped to the flow cell at 10 ml h−1 for 3 h to facilitate cell attachment to the glass slide surface. Culture medium was replaced with 200 ml of fresh TMM containing the corresponding antibiotics and inducers and then fed to the flow cell at the same rate for 15 h. After incubation, the flow cell unit was disconnected and blocked (never allowing it to dry). It was placed at room temperature and the SYTO9 mix was added using a 5 ml syringe. The flow cell was incubated with SYTO9 in the dark for 20 min at room temperature. Eight images in random positions were taken with an Olympus FV100 confocal microscope using a long working distance × 40 Olympus LWDCDPLAN 40LP lens. 3D images of each spot were obtained using IMARIS software, and quantitative analysis of biofilm was performed using COMSTAT software. For the SYTO9 stain preparation, the LIVE/DEAD BacLight Bacterial Viability Kit L7007 was used. Three millilitre of dye was prepared in a 30 ml glass beaker mixing; 7.5 μl of component A (SYTO 9 dye, 1.67 mM / propidium iodide, 1.67 mM), 7.5 μl of component B (SYTO 9 dye, 1.67 mM / propidium iodide, 18.3 mM) and 3 ml of TMM.
Mutations in YmoB (cysteine to serine, phenylalanine to leucine, phenylalanine to tyrosine) were introduced using the QuickChange site directed mutagenesis Kit (Stratagene) with the appropriate complementary mutagenic primers. We confirmed the constructs by DNA sequencing.
[C117S]YmoB sample preparation for NMR experiments.13C/15N-labelled [C117S]YmoB variant was expressed in E. coli BL21 (DE3) in minimal medium with 15NH4Cl and/D-[13C] glucose as the sole sources of nitrogen and carbon respectively39. Cells were lysed by sonication. Soluble protein was purified by His-Tag affinity chromatography (Ni-NTA agarose resin QIAGEN) and samples were eluted using buffer A (20 mM Trizma hydrochloride, 800 mM NaCl, 400 mM imidazole, 5 mM DTT and pH=8.00). Protein was further purified by SEC using HiLoad Superdex 75 prepgrade columns (GE Healthcare) and buffer B (pH=7.00 buffered with 20 mM [NaH2PO4+Na2HPO4], 150 mM NaCl, 1 mM tris-carboxyethylphosphine (TCEP), 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 0.01% w/v NaN3). Fractions containing the purified protein were concentrated using a centrifugal concentrator VIVASPIN 20 10,000 MWCO PES to the mentioned concentrations for NMR experiments.
NMR acquisition, processing and analysis. NMR spectra listed in Supplementary Table 2 were acquired at 1.4 mM protein concentration, in buffer A and 25 °C on 600 or 800 MHz Bruker spectrometers equipped with a cryo-pulse-field gradient triple-resonance probe. NMR spectra listed at Supplementary Table 3 were acquired at 1.0 mM protein concentration in buffer B and 25 °C on 900 or 800 MHz Agilent spectrometers equipped with a cryo- (900 MHz) and room temperature (800 MHz) pulse-field gradient triple- resonance probes. Hydrogen bonds were identified from a H/D exchange experiment. A lyophilized sample of 1.4 mM [C117S] YmoB was suspended in D2O reaching the same concentration, and a two-dimensional 1H-15N-heteronuclear single quantum correlation (HSQC) experiment was acquired after 4 min of sample preparation. Stereospecific assignments of valine and leucine methyl groups were obtained from a constant time 1H–13C HSQC on a 10% 13C/100% 15N [C117S]YmoB 1.4 mM sample in buffer B. One fresh sample of 15N–13C labelled [C117S]YmoB at 1.0 mM in buffer B was used for NOE correlation spectroscopy (NOESY) experiments and an equivalent fresh sample for total correlation spectroscopy (TOCSY) experiments. Protein distance restraints were obtained from 3D 1H–15N edited NOESY HSQC, 3D 1H–13C edited NOESY (aromatic optimized in D2O), and 3D 1H–13C edited NOESY HSQC in D2O experiments. NOESY and TOCSY spectra were recorded using gradient sensitivity-enhanced pulse sequences from the BioPack library (Agilent Inc.). Mixing time in NOESY experiments was set to 100 ms and to 14 ms in TOCSY experiments. The non-uniform sampling (NUS) schedules were prepared by program nussampler from the MDDNMR software package40. When appropriate, the sampling probability density was biased for aliphatic 13C-aliphatic evolution according to the single-bond 13C–13C homonuclear coupling constant of 35 Hz. When appropriate, sampling was exponentially weighted according to the maximum evolution time. Data processing and analysis were carried out with MDDNMR, NMRPipe41, NMRViewJ42 and CARA43. Proton chemical shifts were referenced using 4,4-dimethyl-4-silapentane-1-sulfonic acid as an internal standard, whereas 15N and 13C chemical shifts were indirectly referenced.
Structure determination. NOE distance restraints were obtained iteratively using the Unio’10/CYANA 2.1 (refs 27, 28, 29) suite program and manually inspected. Structure was determined by simulated annealing using the torsion angle dynamic simulation program CYANA 2.1 and further water refinement with CNS 1.2.1 (refs 30, 31). Protein structure calculation was based on Unio’10/CYANA generated upper distances using only unambiguously assigned restraints derived from NOESY experiments, hydrogen bond restraints based on H/D 1H-15N-HSQC exchange experiments and dihedral angle restraints calculated with TALOS+ (ref. 44). An ensemble of 100 protein structures was generated and the 20 lowest energy models were checked using PROCHECK-NMR45. None of the structures contained distance or dihedral angle violations >0.5 Å or 5°, respectively. Inter-helical angles were measured with Chimera v10.1 (ref. 46). Ramachandran analysis is as follows: 93.1% most favoured regions, 6.3% additional allowed regions, 0.5% generously allowed regions and 2.2% disallowed regions. Molecular images were generated with Chimera, Gimp (http://www.gimp.org/) and InkScape (https://inkscape.org/en/). Pocket identification was carried out with DogSiteScorer server33 using default settings. The TomB homology model was built using Modeller34 starting with YmoB as template structure and only rebuilding the regions surrounding insertions or deletions in the sequence alignment. Logo representation was generated using web service from http://weblogo.threeplusone.com47 and manually coloured using InkScape.
15N-labelled [C117S]YmoB, Hha and YmoA proteins were prepared as previously described for 13C/15N-labelled [C117S]YmoB. For MTSL-protein functionalization, reductant was firstly removed using PD10 desalting column (GE). Commercially available MTSL tag (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate from Santa Cruz Biotechnology) was dissolved in acetone and 10 molar equivalents of MTSL were added to the protein sample and left reacting overnight at 4 °C under stirring conditions. Excess of MTSL was removed using P10 desalting column in buffer containing 20 mM [NaH2PO4+Na2HPO4] pH=7, 150 mM NaCl, 0.1 mM EDTA. Protein samples were prepared fresh and measured immediately after preparation. NMR experiments were recorded at 298 K with a 600 MHz Bruker Avance III spectrometer equipped with a TCI cryo-probe. NMR spectra were analysed using CCPNMR Analysis48.
Hha oxidation by [C117S]YmoB. A solution containing 500 μM Hha and 500 μM [C117S]YmoB was incubated for 30 min in 20 mM [NaH2PO4+Na2HPO4], 150 mM NaCl, 0.1 mM EDTA pH=7.00. Parallel experiments were carried out with the same batch of Hha with [F111L,C117S]YmoB, [F111Y, C117S]YmoB or without any YmoB variant. Hha and YmoB were separated by SEC in 50 mM ammonium acetate buffer. Hha was digested with trypsin and the resulting peptides were analysed by nanoHPLC‐ESI‐MS/MS (nano high-performance liquid chromatography coupled to tandem mass spectrometry with nanoelectrospray ionization) in a LTQ‐FT Ultra (Thermo Scientific) mass spectrometer and processed and analysed with Xcalibur software (versus 2.02 SR2). Ion deconvolution to zero charged monoisotopic masses was performed using Xtract algorithm in Xcalibur software.
The relative abundance of the oxidized species was estimated from the ratio of the peptide spectrum matches (PSM) corresponding to peptides with oxidized cysteine (sulfenic, sulfinic or sulfonic acids) with respect to the PSM number of all cysteine-containing peptides. The incubation and digestion steps were carried out at least in duplicate and at least two separate samples of each peptide mixture were analysed by mass-spectrometry.
In a separate experiment the relative abundance of oxidized peptides was also calculated considering the chromatographic peak areas of the corresponding peptides using Skyline 3.1 software49, obtaining equivalent results (data not shown).
YmoB or [C117S]YmoB at 200 μM protein concentration and YmoA alone or in the presence of [C117S]YmoB were incubated in the presence of air for 12 days in a buffered solution of 20 mM [NaH2PO4+Na2HPO4] pH=7.0, 150 mM NaCl, 0.1 mM EDTA and 0.01% w/v NaN3) with or without DTT (2 mM) and in the presence of dimedone in a 1:10 ratio. Peptides were generated with endoproteinase GluC digestion and analysed as described above.
Toxicity tests of Hha and TomB variants.
Overnight cultures of E. coli BW25113 Δhha harbouring pCA24N, pCA24N-hha, pCA24N-hha(C18E) or pCA24N-hha (C18I) were inoculated into 25 ml of LB medium with 30 μg ml−1 chloramphenicol and 1 mM IPTG at an OD600 of 0.04. For TomB and [E53K]TomB, overnight cultures of E. coli ATCC25404 Δhha ΔtomB harbouring pCA24N-tomB-hha or pCA24N-tomB(E53K)-hha were inoculated into 25 ml of LB medium with 30 μg ml−1 chloramphenicol at an OD600 of 0.05. After an hour of inoculation, 0.1 mM IPTG was added. Growth was evaluated periodically by measuring OD600. For each strain, two or three independent cultures were evaluated. To check total protein expression, cultures at an OD600 of 0.5 were induced with 1 mM IPTG for 4 h. Cells were sonicated, centrifuged, and 25 μg of cell lysates (supernatant) were resolved via 18% SDS–polyacrylamide gel electroporesis. Total protein of cell lysates was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA).
The hha(C18E) mutation was generated in a pET15b expression plasmid35 that produces a His6-tagged form of Hha. The 15N-labelled wild type and [C18E] proteins were purified by His-Tag affinity chromatography (Ni-NTA agarose resin QIAGEN) by eluting with 20 mM Trizma hydrochloride, 800 mM NaCl, 400 mM imidazole, 5 mM DTT and pH=8.00. A final purification step on a Superdex 75 column in 20 mM sodium phosphate, 150 mM NaCl, 1 mM TCEP, 0.01% w/v NaN3, pH=7.00 yielded pure proteins as monomers.
Before H2O2 treatment, TCEP was removed by passing the protein solution through a PD-10 column. After measuring the reference NMR spectra, the wild-type protein was treated with 5 mM H2O2 and spectra were recorded every 130 min.
All relevant data are available from the authors. NMR assignment and the structural models have been deposited in the RCSB PDB with accession numbers RCSB104115 and 2MN2.
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We thank Cristina Madrid and Antonio Juarez for helpful discussions and for providing plasmids and Laura Villarreal and Mar Vilanova for help in MS experiments and Laareb Irrem Mohamad for help in DLS experiments. This work was partially supported by funds from the Spanish MINECO (BIO2010-15683 and BIO2013-45793R –cofounded with European Structural Funds, FEDER-), the EC FP7 BioNMR project (contract 261863) and the ARO (W911NF-14-1-0279). T.K.W. is the Biotechnology Endowed Professor at the Pennsylvania State University. NMR experiments were performed at the ICTS NMR LRB and CCITUB (University of Barcelona) and the Swedish NMR Centre (Goteborg University). MS experiments were performed at IRB Barcelona MS Core Facility (member of the BMBS COST Action BM 1403 and Proteored, PRB2-ISCIII (IPT13/0001—ISCIII-SGEFI / ERDF). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). Development of Chimera package by UCSF is supported by NIGMS P41-GM103311.
O.M. performed and analysed structural, functional and mass spectrometry experiments. J.M.C.T, V.W.C.S. T.L.W. and J.G. prepared and analysed mutants. A.M. performed oxygen dependency experiments. J.M.C.T., M.G. and M.V. performed and analysed mass spectrometry experiments. J.M.C.T. performed experiments leading to the identification of the Hha binding site. T.N.C. and I.A. contributed to NMR experiments and structural analysis. M.M. and V.Y.O. performed interleaved-NUS NMR experiments. J.G. and T.N.C. screened and optimized constructs and NMR conditions. T.K.W. designed and analysed functional experiments. M.P. conceived and supervised the project and wrote the paper with contributions from all other co-authors.
Correspondence to Thomas K. Wood or Miquel Pons.
Supplementary Figures 1-10 and Supplementary Tables 1-4.


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The federal government is liable to Moda Health Plan, Inc. for failing to make risk corridor payments totaling $214 million, a federal judge ruled last week.
The risk corridors program is a risk-reduction mechanism established by the Affordable Care Act (ACA) to encourage insurer participation in new initiatives. Because new initiatives (such as federally funded health exchanges) lacked historical data necessary for underwriting, the risk-reduction mechanisms protected early adopters from significant losses. The program provided for government reimbursement of certain losses for qualified health plans on the health exchanges, but required profitable health plans to pay excess gains to the government.
A Recent Risk Corridor Litigation Success: Moda Health Plan, Inc. v. U.S.
Moda Health Plan, Inc. (Moda) suffered $214 million in losses after being paid only 12.6 percent of its claimed risk corridor payments for 2014 and nothing for 2015. To recover the outstanding balance of its risk corridor payments, Moda sued the government. Moda argued that the government was obligated under the ACA to make risk corridor payments or, in the alternative, that the risk corridors program was an implied-in-fact contract between the insurers and the government. The government denied these allegations, largely relying on Congress’ failure to appropriate money for the program and its declaration in the appropriations bill that the risk corridors program be budget-neutral.
In Contrast, the Unsuccessful Land of Lincoln Mut. Health Ins. Co. v. U.S.
Judge Wheeler’s decision contrasts with another recent decision in the Court of Federal Claims: Land of Lincoln Mut. Health Ins. Co. v. U.S.4 In Land of Lincoln, a different judge – Judge Lettow – denied Land of Lincoln’s claim for risk corridor payments. Judge Lettow held that the ACA was ambiguous regarding the government’s responsibility for deficits or excess funds in the risk corridors program and, further, risk corridor payments were not required by express or implied contract with the government. Congress issued its declaration that the risk corridors program be budget-neutral before the 2015 plan year, and according to Judge Lettow’s analysis, it was not reasonable for an insurer to expect to be paid the full risk corridors obligations in 2015.
Whether under statute or contract, the Court finds that the Government made a promise in the risk corridors program that it has yet to fulfill. Today, the Court directs the Government to fulfill that promise. After all, “to say to [Moda], ‘The joke is on you. You shouldn’t have trusted us,’ is hardly worthy of our great government.” Brandt v. Hickel, 427 F.2d 53, 57 (9th Cir. 1970).
Several similar cases remain pending, including cases brought by Health Republic Insurance Company, Highmark and CoOpportunity Health. Although the government indicated a willingness to settle the matters early in litigation, it has been vigilantly defending them. In addition to financial considerations, political factors may affect the decision to defend or settle these cases. On one hand, the Trump administration has sharply criticized the law it is defending in these cases, but on the other hand, settling the claims could be viewed as a bailout for the insurance companies. Still, bailout may be more desirable than the risk of market collapse associated with nonpayment. Many insurers have faced significant adverse results from the uncertainty around the risk corridors program.
As more decisions are issued in these cases, insurers should get a clearer idea of the likelihood of government payouts. The U.S. Court of Appeals for the Federal Circuit may weigh in to resolve the conflicting guidance from the Court of Federal Claims, as Land of Lincoln appealed its unfavorable decision and the government is likely to appeal the Moda decision. Until then, Stradley’s Insurance Practice Group is available to answer any questions about the risk corridors program, the ACA or other regulatory matters.
1 Centers for Medicare & Medicaid Services, Risk Corridor Payment for the 2014 Benefit Year (Nov. 19, 2015), available at https://www.cms.gov/CCIIO/Resources/Regulations-and-Guidance/Downloads/RC_Obligation_Guidance_11-19-15.pdf.
2 Centers for Medicare & Medicaid Services, Risk Corridor Payment and Charge Amounts for the 2015 Benefit Year (Nov. 18, 2016), available at https://www.cms.gov/CCIIO/Resources/Regulations-and-Guidance/Downloads/2015-RC-Issuer-level-Report-11-18-16-FINAL-v2.pdf.
3 Moda Health Plan, Inc. v. U.S., No. 16-649C, 2017 WL 527588 (Fed. Cl. Feb. 9, 2017).
4 Land of Lincoln Mut. Health Ins. Co. v. U.S., 129 Fed. Cl. 81 (2016).
Stradley Ronon received top marks in several practice areas in the 2017 U.S. News – Best Lawyers “Best Law Firms” publication, including 13 national rankings and 13 top-tier rankings in Philadelphia. The firm’s insurance law practice group was one of the practice areas ranked both nationally and in the top tier in Philadelphia.
Sam Arena has been elected to a two-year term as a member of the governing body of the American Bar Association Tort Trial & Insurance Practice Section (TIPS), TIPS Section Council.
Sam and Nicole Stover co-authored a chapter in the Fourth Edition of Financial Institution Bonds, published by the American Bar Association. The chapter, titled “Insuring Agreement (B): On Premises Coverage,” provides analysis and commentary regarding the On Premises coverage found in the Surety & Fidelity Association of America’s Financial Institution Bond and the Insurance Services Office’s Financial Institution Crime Policy.
Twenty-three Stradley Ronon attorneys were named to the recently released 2017 edition of The Best Lawyers in America, regarded as a definitive guide to legal excellence. Best Lawyers lists are compiled based on an exhaustive peer-review evaluation. Sam Arena and Steve Davis from the insurance practice group were included on this list.
Steve Davis moderated a panel entitled, “Insurance Department General Counsel Discussion,” at the Property Casualty Insurers Association of America’s Northeast General Counsel Seminar in Philadelphia.
Jana Landon was part of the panel at the AON Financial Services Group National Conference discussing cybersecurity litigation trends. She was also a panelist for, “Attorney Ethics/Social Media/Data Privacy,” at the Eastern District of Pennsylvania Bankruptcy Conference in Philadelphia. Jana was also part of the panel, “Evolving Cyber Threats,” at the Association of Insurance Compliance Professionals Annual Conference in Orlando, Florida. Jana was a featured speaker at the Chamber of Commerce of Southern New Jersey’s “Cybersecurity Workshop” in Voorhees, New Jersey.
Craig Blackman and Jana presented the seminar, “How to Protect Your Nonprofit from Cyber Attacks,” in Philadelphia. The session was moderated by Catherine M. Ward.
Stradley Ronon was honored as the top litigation department among Pennsylvania’s midsize law firms in The Legal Intelligencer’s “Legal & Litigation Departments of the Year” supplement. The publication chose the winners “based on the big victories and important cases that demonstrated why clients chose them when litigation was on the table.” Stradley Ronon was praised for its “emphasis on efficiency” and ability to determine the issues most important to a client.
Jana Landon is moderating a cybersecurity panel entitled Beyond Breach: Everything You Need to Know About Cyber on March 23 at Philly I-Day. Philly I-Day is the Delaware Valley’s premier educational and networking event for insurance and risk management professionals.

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The Internet has revolutionized communication throughout the world, allowing people to correspond instantaneously at relatively low cost. Federal Judge Stewart Dalzell called the Internet the “most participatory form of mass speech yet developed.” However, this speech-enhancing medium has led to numerous controversies, causing many people to view the Internet as the premier First Amendment battleground.
Many Internet free-speech battles concern laws that restrict content, such as the Communications Decency Act and the Child Online Protection Act, and laws that mandate Internet filtering in public libraries or schools, such as the Children’s Internet Protection Act.
But the Internet has presented another, pressing First Amendment question unique to public schools: Under what circumstances may school officials punish students for the content of their online expression? In its famous 1969 decision Tinker v. Des Moines Independent Community School District, the U.S. Supreme Court wrote that students do not shed their free-expression rights “at the schoolhouse gate.” In more recent times, the concern has become whether school officials can, consistent with the First Amendment, extend their authority from the schoolhouse gate to students’ personal computers. Some school officials have extended their authority by punishing students for online speech even though students created the speech off campus. In many cases, students have turned to the Internet to express themselves about principals, teachers and other students.
One threshold question is whether the student cyberspeech in question is a true threat. Under First Amendment law, true threats do not receive First Amendment protection. The Supreme Court defined true threats in the cross-burning decision Virginia v. Black as follows: “‘True threats' encompass those statements where the speaker means to communicate a serious expression of an intent to commit an act of unlawful violence to a particular individual or group of individuals."
The lower courts have developed different tests, but many ask whether an objective, rational recipient of the statement would reasonably believe it to be a threat.
Another form of online expression that may not cross the line into true threats is cyberbullying — where one student harasses or intimidates another student online. Some but not all cyberbullying could qualify as a true threat. State legislators and school officials have addressed the problem by passing laws or policies to punish students who engage in such behavior. Many states have passed laws that require school districts to adopt policies that address cyberbullying.
One potential problem is that a broad definition of cyberbullying could include almost any online or electronic student speech that another student finds offensive. It will take litigation for this area of the law to develop.
Cases outside the online arena provide guidance. For instance, a federal district court in Maine ruled in Klein v. Smith (1986) that school officials violated the constitutional rights of a student when they suspended him for gesturing at a teacher with his middle finger raised at an off-campus restaurant. The judge determined that the student’s disrespectful act was “too attenuated” with school functions to be punishable by school officials.
Sometimes, school officials may argue for even greater control over student Internet speech under the Supreme Court’s decision in Hazelwood School District v. Kuhlmeier (1988). In Hazelwood, the Supreme Court ruled that Hazelwood school officials could censor articles on teen pregnancy and divorce in the student newspaper. The Court established the Hazelwood standard, which provides that school officials can regulate school-sponsored student speech if they have a reasonable educational reason for doing so. For example, if a student created a Web site on school grounds using school computers during a class, the school would have broad authority under Hazelwood to silence the expression and punish the student.
* Was the content created as part of the school curriculum, such as a class project or school newspaper?
* Was the content created on school computers?
* Was the content distributed (particularly by the student who wrote the material) at school?
If the student created the material as part of the school curriculum, school officials could apply the Hazelwood standard. If the content was created on school computers, Hazelwood might apply. If the content was created off campus but distributed by the student on school grounds, most courts would apply the Tinker standard and some might apply the Fraser standard.
Students generally have broad freedom to express themselves on the Internet on their own time, using off-campus computers. However, some school officials have suspended students for their off-campus Web postings that lampooned or criticized school officials or contained vulgar commentary.
Some courts have sided with the students, saying that school officials may not censor student speech unless they can reasonably forecast that the speech will cause a substantial disruption of the school environment or invade the rights of others. Other courts and commentators have said that school officials simply lack the authority to regulate students’ off-campus behavior — on or off the Internet.
The lower courts certainly have decided the student Internet speech cases differently. For example, a federal court in Missouri ruled in Beussink v. Woodland R-IV School District (1998) that school officials violated the First Amendment rights of a student when they suspended him for 10 days for his home page that criticized the school. The case involved student Brandon Beussink who created a Web page at his home that used vulgar language to criticize the principal, teachers and other aspects of the school environment.
Beussink did not use school computers to create his Web page, though he did access his home page from computers in the school library. The school principal suspended Beussink because he was upset at the content of the Web page. After he was suspended, Beussink sued alleging a violation of his First Amendment rights. A federal judge agreed, finding that the principal committed a legal error in punishing Beussink simply because he disliked the content of the home page.
School officials expelled Swidler, citing the extreme emotional distress suffered by the math teacher and the disruption the Web site caused at the school. Swidler argued in a lawsuit that his Web page was a form of protected speech.
The high court then determined whether school officials had the authority to regulate the student’s Web site. Swidler argued that the Web site was beyond school officials’ control because he created it off-campus. The court disagreed, writing: “We find there is a sufficient nexus between the web site and the school campus to consider the speech as occurring on-campus.” The court determined the speech occurred on campus because the student accessed the site at school, showed it to a fellow student and informed other students of the site. “We hold that where speech that is aimed at a specific school and/or its personnel is brought onto the school campus or accessed at school by its originator, the speech will be considered on-campus speech,” the court wrote.
The court then reasoned that school officials could punish Swidler under the Fraser and Tinker standards. The school could punish Swidler under Fraser because the Web site was vulgar and highly offensive. It could punish him under Tinker because the Web site caused a substantial disruption of school activities.
Most courts have applied the Tinker “substantial disruption” test to student online speech cases. For example, the 2nd Circuit ruled in May 2008 in Doninger v. Niehoff that school officials could punish a student for blogging critical comments about a school administrator. "We have determined, however, that a student may be disciplined for expressive conduct, even conduct occurring off school grounds, when this conduct 'would foreseeably create a risk of substantial disruption within the school environment,' or at least when it was similarly foreseeable that the off-campus expression might also reach campus," the court concluded.
* Whether school officials have any legal authority to regulate student online expression created off-campus.
* Whether and under what standard school officials can regulate off-campus student speech that is distributed at school either by the student who created it or other students.
* Whether school officials have more authority to regulate student online speech if it is created off-campus but contains a link to the school’s own Web site and is aimed directly at the school audience.
It will probably take a decision by the U.S. Supreme Court to provide the necessary guidance to resolve these thorny issues of student cyberspeech.


The Texas Compassionate Use Act (Senate Bill 339) was enacted by the Texas Legislature in 2015 (84th Legislative Session). The bill required DPS create a secure registry of physicians who treat epilepsy for the purpose of prescribing low-THC cannabis to patients who have been diagnosed with intractable epilepsy. In addition, the bill required DPS to license at least three dispensing organizations by Sept. 1, 2017, should they meet the requirements. The license authorizes the organizations to cultivate, process and dispense low-THC cannabis to prescribed patients.
As of December 15, 2017, the department has issued three dispensing organization licenses: Cansortium Texas was licensed on September 1, 2017; Compassionate Cultivation was licensed on October 31, 2017; and Surterra Texas was licensed on December 15, 2017.
Contact information for Cansortium Texas, Compassionate Cultivation, and Surterra Texas can be found on the FAQ tab under Dispensing Organization.
The department has issued licenses to three dispensing organizations and is currently accepting registration applications from physicians. List of registered physicians.
Once a prescription is dispensed, the product will be labeled with information to assist law enforcement in confirming the legitimacy of the prescription and the patient's legal right to possess low-THC cannabis.
Law Enforcement can call the Department to verify a patient or dispensing organization employee's status.
1. Who currently holds a license as a dispensing organization?
2. What is "low-THC cannabis"?
Not less than 10 percent by weight of cannabidiol.
3. What medical conditions are covered under this program?
The Compassionate Use Program is statutorily limited to patients in Texas with intractable epilepsy. Adding conditions will require legislative action.
4. Why did DPS conditionally approve only three licenses? Will the number of licenses be increased in the future?
This was based on an analysis of other states' compassionate–use programs, the number of patients in Texas with intractable epilepsy, and statutory requirements. Texas Health and Safety Code Chapter 487 requires that the department issue at least three licenses but no more than the number of licenses necessary to ensure reasonable statewide access to – and availability of – low-THC cannabis for patients prescribed low-THC cannabis under Chapter 169, Occupations Code. A decision to increase the number will be made if and when it is determined that more licenses are required in order to ensure reasonable statewide access.
5. How will the dispensing organizations ensure state wide access for patients?
The dispensing organizations' employees will be delivering prescriptions, as well as potentially filling prescriptions over the counter. You may wish to contact the conditionally approved dispensing organizations to determine their specific delivery approach.
6. Will patients be able to grow their own cannabis?
No. Only licensed dispensers will be able grow cannabis and only for use in the production of low-THC cannabis. Patients are required to purchase low-THC cannabis products from a licensed dispensing organization.
7. Will it be legal for patients to smoke low-THC cannabis?
No. Texas Occupations Code §169.001 specifically excludes smoking from the definition of "medical use.".
8. What protections will patients and legal guardians have from criminal prosecution?
Texas Health and Safety Code §481.111(e)(1) provides exemptions from state laws prohibiting possession of marijuana for patients (and their legal guardians) for whom low-THC cannabis is prescribed under a valid prescription from a dispensing organization.
9. Will dispensing organizations be allowed to make products from different plant strains even if the plant has more THC, as long as the end product meets statutory levels?
Texas Health & Safety Code §481.111(e)(2) provides the statutory guidelines for what dispensing organizations are legally authorized to possess.
10. Where do I send a complaint on a dispensing organization or physician?
Complaints regarding dispensing organizations may be submitted through the Compassionate Use Program Contact Us or by mail. For complaints regarding Physicians, please contact their respective medical board.
1. Which medical conditions are approved for low-THC cannabis?
The Texas Compassionate Use Act specifically limits the use of low-THC cannabis to patients with intractable epilepsy.
2. How does a patient qualify for the program?
A second qualified physician has concurred with the determination.
3. Will patients have to register with the state or pay a fee?
No. Statute does not require patients to register or pay a fee. Patient information will be retained in the Compassionate Use Registry. A qualified physician will enter a patient's information into the Compassionate Use Registry.
4. Are there any age restrictions for "Low-THC Cannabis" prescriptions?
Statute places no limitations on the age of the patient. Patients under 18 may require a legal guardian.
5. Will I be able to bring my child from out-of-state to acquire low-THC cannabis in Texas?
Patients must be permanent Texas residents.
6. How much will low-THC cannabis cost?
Prices will be set by the licensed dispensing organizations, based on the market. DPS does not regulate the cost of the product.
7. Will patients' legal guardians be able to pick up the order from a dispensing organization?
Yes. Physicians will be required to document the names and last 5 of SSN of legal guardians in the Compassionate Use Registry. Patients or legal guardians will be required to show proof of identification to the dispensing organization.
8. Can you have a prescription from another state filled in Texas?
No, Texas Health and Safety Code §487.107 only authorizes the possession of low-THC cannabis that is obtained through a prescription issued by a physician registered with the CUP program and the dispensing of a prescription for a person listed as a patient in the compassionate-use registry.
1.	How many dispensing organizations will be licensed in Texas?
Texas Health and Safety Code Ch 487 requires the department license at least three dispensing organizations, provided at least three applicants have met the statutory requirements.
2.	Will testing laboratories be licensed or approved by the state?
Texas Health and Safety Code Ch.487 regulates the cultivation, processing, and dispensing of low-THC cannabis and does not specifically address the licensing of low-THC cannabis testing laboratories. Only licensed dispensing organizations may test low-THC products.
3.	Will I need a registration to work at a dispensing organization?
Yes. Texas Health & Safety Code §487.151 provides the requirements for employees to obtain a registration to work at a dispensing organization. Additional information can be found in the Administrative rules, Section 12.12.
4. Are there restrictions on dispensing organization proximity to schools or daycares?
(n) All regulated premises shall be located at least 1000 feet from any private or public school or day care center that existed prior to the date of initial license application, measured from the closest points on the respective property lines.
5. Can local governments prohibit the operations of a licensed dispensing organization?
Texas Health and Safety Code Sec. 487.201 COUNTIES AND MUNICIPALITIES MAY NOT PROHIBIT LOW-THC CANNABIS. A municipality, county, or other political subdivision may not enact, adopt, or enforce a rule, ordinance, order, resolution, or other regulation that prohibits the cultivation, production, dispensing, or possession of low-THC cannabis, as authorized by this chapter.
6. What is the contact information for the three licensed dispensing organizations?
1. What are the qualifications for physicians to prescribe low-THC cannabis?
2. As a physician, I am concerned about providing a prescription for low-THC cannabis. Will the term "prescription" be changed to "recommendation?"
1.	What is the Compassionate Use Registry of Texas (CURT)?
The Compassionate Use Registry of Texas (CURT) is the systems that allow physicians to register and prescribe "Low-THC" cannabis to patients with intractable epilepsy. Once prescribed, dispensing organizations search the patient in the system and dispense the medication.
2.	Who needs to register in CURT?
Physicians who qualify under Texas Compassionate Use Act §169.002 and would like to prescribe "Low-THC" cannabis for qualifying patients will need to register through CURT.
3.	Is CURT available to physicians, dispensing organizations and law enforcement 24hrs a day?
Yes, system is available to physicians and dispensing organizations 24 hours a day. Law Enforcement can call the Department to verify a patient or dispensing organization employee's status.
1. What should a physician do if the application for registration is denied?
Contact Regulatory Services Division for more information regarding the denial.
2. Does my patient need to see a secondary physician for the secondary consult in CURT?
No. It is not necessary for the secondary physician to have contact with the patient. The primary physician can discuss the patient's condition with secondary physician for approval.
3. What information is needed from the secondary physician for a consult?
The secondary physician will need to provide their CURT username to the primary physician. The primary physician will enter this information under the patient details.
4. What should I do if the patient I am trying to add is already associated with another physician?
The patient will need to contact their previous physician and ask to have the relationship terminated.
1. Do I need to register in CURT?
Patients do not need to register in CURT, nor will they need access to CURT. The physician will add the patient into CURT.
2. How do I get my prescription?
The physician will enter the prescription in the CURT system. Patient or legal guardian will then have the ability to go to any of the dispensaries to have the prescription filled. Patient or legal guardian will need to provide identification and patient's last name, date of birth, and last 5 of social security number.
3. What should I do if my prescription is not found by the dispensing organization?
Ensure dispensing organization entered the information correct. If so, contact your doctor to ensure they entered the prescription information correctly.
1. How do employees register in CURT?
Dispensing Organizations will need to submit RSD-52s for all employees. Once they are registered, the employee will be emailed a link to create their account in CURT.


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Bammert v. Don's Super Valu, Inc.
Home > Case Law > Employment > Bammert v. Don's Super Valu, Inc.
At-will employees, terminated in retaliation, wrongful discharge action, public policy exception.
Joint employers, WARN Act violations, ordered a closing or layoff.
Bard v. Bath Iron Works Corp.
Retaliatory discharge, Whistleblowers Protection Act, threaten discriminate against an employee.
Non-compete agreements enforced by injunction, employee agreement, geographical scope and duration are reasonable.
Employer-administered psychological examinations, unconstitutional searches, The Fourth Amendment, highly personal private information, state action.
Available for work, qualified, willing, able, and ready to accept employment, no good cause to refuse.
Intentional representations, future of plan benefits, act of plan administration giving rise to a fiduciary duty, fiduciary receiver.
Williams v. Mohawk Industries., Inc.
Civil RICO claim, pattern of racketeering activity, Racketeer Influenced and Corrupt Organizations Act.
Starbucks Corp. v. Superior Court; A disclaimer in a standard job application advising an applicant that he or she need not complete certain sections as provided by local law must be clear and conspicuous .
Harrison v. Benchmark Electronics Huntsville, Inc. Under the Americans Disabilities Act an employer may not conduct pre-offer medical examination or make inquiries of job applicant whether applicant.
Gross v. Hale-Halsell Co. An employer may order a plant closing or mass layoff before the conclusion of the statutory sixty-day notice period if the closing or mass layoff is caused by business circumstances that were not reasonably foreseeable.
LaRue v. DeWolff, Boberg & Associates, Inc.; Although no remedy is available for individual injuries in defined contribution plan, individuals may recover for fiduciary breaches that impair the value of plan assets in the participant's individual account.
Bank of New York Mellon v. Realogy Corporation; the fact that loans under credit agreements are usually funded in cash does not mean that the word "loan" cannot encompass borrowings funded otherwise.

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