gram - positive bacteria , 
 particularly of the bacillus and clostridium genera , have a sporulation mechanism 
 by which they can protect their dna and become dormant under harsh 
 conditions that would otherwise kill them . this survival mechanism allows for the bacteria to survive in a 
 metabolically inactive state for long periods of time until conditions , 
 such as temperature or nutrient content , allow for germination and 
 outgrowth . some spores have been recovered and cultured after storage 
 in amber for millions of years . the structure 
 of the spore is well - understood , separated into distinct regions : 
 an outer spore coat which is laminar and serves as a protective barrier ; the cortex , composed of cross - linked peptidoglycan distinct from 
 that found in the germinated bacterial cell wall and implicated in 
 the heat resistance of spores ; and the core , containing the bacterial dna as well 
 as protective molecules such as small acid - soluble proteins ( sasps ) and an aquo - coordination complex between ca and dipicolinic 
 acid ( pyridine-2,6-dicarboxylic acid ) . the spore core has a lowered 
 water content compared to that of vegetative cells , which is thought 
 to be a major factor in spore heat resistance . the exact 
 nature of the water in the spore core is not well understood . one theory is 
 that the core water is in an amorphous ( glassy ) state . this was suggested 
 by sapru and labuza in 1993 by predicting 
 glass transition temperatures for spores based on inactivation kinetics 
 and finding that more heat - resistant spores had higher predicted glass 
 transition temperatures . ablett in 1999 used differential scanning calorimetry to identify features of b. subtilis spores that were indicative of low moisture 
 content even in an excess water environment and using c nmr on calcium dipicolinate concluded it was likely in an amorphous 
 solid state indicative of a core with low water content consistent 
 with a glassy state . the other theory is that the core water is in a gel - like state . this hypothesis was put forward initially by black and gerhardt as a means of explaining their results on water 
 uptake in spores using tritium - labeled h2o ; they proposed 
 that the spore core is an insoluble gel whose components are cross - linked 
 but water - permeable . more recently , some have argued for the gel - like core theory based on deuterium relaxation 
 times recorded from d2o - exchanged spores . proponents of 
 the gel core theory assert that the gel provides thermal heat resistance 
 during dormancy by protecting proteins against thermal denaturation , 
 assisted by the lower water content of the core . sunde et al . asserted that , 
 were the core in a glassy state , there would be a broad signal apparent 
 in a h nmr spectrum of the spore ; however , such a signal 
 would be undetectable using the magnetic relaxation dispersion experiments 
 as performed . analyzed 
 a quadrupolar - echo spectrum of hydrated spores and concluded that 
 its line shape represents mobile water and labile deuterons from proteins , 
 nucleic acids , and peptidoglycan . thus , no immobilized water could 
 be detected in the h nmr spectrum of fully hydrated spores . here , we report h nmr spectra 
 of deuterated spores 
 of a wild - type bacillus subtilis ( atcc 6051 ) and 
 the chloramphenicol - resistant strain bacillus subtilis 1a578 . deuterium hydrogen exchange 
 experiments were conducted to examine the permeability of water into 
 the spore and to compare them against calcium dipicolinate trihydrate 
 ( a known spore core component ) and bovine serum albumin ( as a spore 
 protein analogue ) . in addition , variable - temperature ( vt ) nmr experiments 
 were performed to determine how the deuteron mobility varies as the 
 spores are heated and cooled . our results suggest that there is immobile 
 water in the spore core that can be detected using quadrupolar echo 
 deuterium nmr . variable - temperature nmr data does not show a gradual 
 increase in molecular mobility proportional to temperature as would 
 be expected for a spore core containing a gel of highly mobile water . 
 instead , our vt nmr data indicate that deuterated biomolecules and 
 deuterated water retain their anisotropic motion properties at 50 
 c . heating to 100 c does not affect viability , and the 
 nmr spectra show that the proteins remain immobile at this temperature . however , 100 c causes a transition of some deuterons from anisotropic 
 to isotropic motion which we attribute to immobile water associated 
 with protein hydration . these observations support the glassy - state 
 core theory for our samples of dried spore powders . nonetheless , we 
 also observe mobile water in the core as recently reported and it remains possible that an aqueous spore suspension could have 
 a gel - like core . bacillus subtilis 1a578 , a chloramphenicol - resistant 
 strain , was obtained from bacillus genetic 
 stock center ( bgsc , department 
 of chemistry at the ohio state university ) . frozen 
 stock was inoculated overnight in 20 ml aliquots of lb medium , which 
 was used to inoculate 200 ml of lb medium at 1% in a 1 l erlenmeyer 
 flask . the culture was allowed to grow ( 200 rpm @ 37 c ) for 
 24 h , after which it was centrifuged at 10000 g at 
 4 c for 20 min , the supernatant decanted , and the pellet resuspended 
 in a separate 1 l flask with 200 ml of chemically defined sporulation 
 medium ( cdsm , composition included in the supporting 
 information ) and allowed to grow 
 ( 200 rpm @ 37 c ) until the culture exhibited at least 80% spores 
 via phase contrast microscopy ( between 3 and 10 days ) . for partially 
 deuterated spores , cultures were inoculated into 25% d2o lb medium and resuspended in 25% d2o cdsm for sporulation . once the sample had sufficient spores , the culture was spun down , 
 washed 1 with sterile milli - q water , spun down , and resuspended 
 in a centrifuge tube in 20 ml of sterile 50% v / v etoh : h2o . the resuspended spores were shaken at 200 rpm at 25 c for 
 2 h to kill any remaining vegetative cells . the spores were then centrifuged 
 3x ( 10000 g , 4 c , 20 min ) ; the supernatant was 
 decanted and the spores were washed 3x with 20 ml aliquots of sterile 
 milli - q water between each centrifugation . after washing , the spores 
 were resuspended in a fresh 20 ml aliquot of sterile water and stored 
 overnight at 4 c ; the following morning the culture was spun 
 down and resuspended in fresh sterile water . the final step of purification 
 involved delivery of the spores into a sterile centrifuge tube via 
 syringe filtration using 3.1 and 1.2 m glass fiber filters 
 connected in series . the purified spores were centrifuged ( 10000 g , 4 c , 20 min ) , the supernatant was decanted , and 
 the pellet was resuspended and transferred to a glass vial with 99% 
 d2o ( total volume 8 ml ) . the spore suspension was frozen 
 with liquid nitrogen and lyophilized for 2 days to form a fluffy , 
 charged white or off - white powder , which was stored at 20 
 c until use . spores were packed into 5 mm ceramic magic - angle - spinning 
 rotors and sealed with o - ring caps for nmr experiments . for 
 deuterium  hydrogen exchange experiments , the lyophilized 
 deuterated spores were transferred to a sterile 15 ml falcon tube 
 and resuspended in sterile milli - q water . the tube was sealed with 
 a plastic cap , covered in parafilm , and stored at 4 c with occasional 
 shaking for 1 week . the exchanged spores were transferred to a centrifuge 
 tube and spun at 10000 g ( 4 c , 20 min ) . water 
 was decanted , and the wet spores were resuspended with sterile milli - q 
 water to aid their transfer into a glass vial prior to lyophilization 
 as described above . deuterium exchange 
 experiments were prepared in a similar manner . lyophilized spores 
 grown in protonated media were suspended in sterile d2o 
 for 1 week before drying . deuterated amorphous calcium 
 dipicolinate trihydrate was prepared by modifying the procedure of 
 johnson et al . to use 99% d2o . after dissolving the dipicolinic acid and adding calcium hydroxide 
 to a ph between 9 and 10 , the heated solution was allowed to cool 
 to room temperature and then stored in a desiccator for 4 days subsequently , 
 the solution was further dried via lyophilization for 24 h. the resulting 
 powder was packed into an nmr rotor to collect h nmr data . crystalline deuterated cadp was prepared using a modification of 
 the procedure by bailey et al . , replacing 
 deionized water with 99% d2o . after addition of ca(oh)2 , the solution was transferred into several 13 mm od test tubes and 
 stored in a desiccator for 1 month . the crystal structure was determined 
 by x - ray crystallography and found to have the same space group and 
 similar dimensions as previously reported data . details of the x - ray crystallography procedures and results 
 are provided as supporting information . was dissolved in 
 99% d2o ( 100 mg/8.0 ml ) , stirred for 24 h at 4 c , 
 and lyophilized for 5 days before packing in a 5 mm rotor and collecting 
 a deuterium solid - state echo ( ssecho ) spectrum . a second sample was 
 prepared by dissolving 100 mg of bsa in 8 ml of d2o , mixing 
 for 24 h , and drying via lyophilization for 19 days . quadrupolar echo h nmr 
 experiments were collected by placing the filled ceramic rotor into 
 a varian 5 mm wide - line nmr probe and inserting the probe into an 
 oxford 400 mhz ( 9.4 t ) magnet ( h frequency = 61.424 mhz ) . data was collected using a varian unityplus console 
 and vnmr 6.1c software on a sun workstation . the pulse sequence ( ssecho , 
 90x190y2acquire ) 
 used a pulse width of 6 s , recycle delay of 120 s , 1 of 60 s , and 2 of 50 s . deuterium nmr calibrations were performed 
 with malonic acid - d4 and pure d2o samples to optimize the 90 pulse width and verify the appropriate 
 spectral appearance of our calibration standards . the h t1 for immobilized water was found to be 24 s using a sample 
 of frozen d2o ( 40 c ) cooled by a stream of 
 nitrogen gas chilled by a xrii fts refrigeration unit . variable - temperature h nmr experiments were performed by delivering heated dry 
 air to the sample . the sample was held at the desired temperature 
 for 15 min prior to data acquisition . data was collected with the 
 ssecho pulse sequence using a delay time of 120 s for each of the 
 2000 transients ( 66 h data collection time ) . an initial spectrum was 
 collected at 21 c followed by heating to 50 c . a spectrum 
 collected at this temperature was compared to the 21 c spectrum , 
 and the sample was then cooled back to 21 c . another spectrum 
 was collected to see whether changes that occurred , if any , at 50 
 c were reversible . the deuterium quadrupolar 
 echo nmr spectrum of partially deuterated b. subtilis atcc 6051 spores is shown in figure 1a . there 
 are three distinct features that are present in this spectrum : feature 
 i , a sharp singlet peak at 0 hz referenced to a liquid d2o standard ; feature ii , a pake doublet with a separation of approximately 
 32 khz ; and feature iii , another pake doublet with a 120 khz separation . a second deuterium nmr spectrum was collected after the b. 
 subtilis atcc 6051 spores were soaked in h2o ( figure 1b ) . here , signals for features i , ii , and iii are 
 present , although the intensity is lower due to either replacing labile 
 deuterons with protons or sample loss after repacking the postexchange 
 spores . after correcting for the latter through scaling of the spectrum 
 by the same proportion as sample lost , an overlay of the pre- and 
 postexchange spectra ( figure 1c ) shows a diminution 
 of features i and ii while feature iii is unchanged . h solid - state 
 nmr spectrum of 25% d2o - labeled b. subtilis atcc 6051 spores ( a ) ; of the same sample after 
 one - week s exchange with h2o ( b ) ; a and b overlaid 
 on the same axis and scaled for sample loss ( c ) . three main features 
 ( i , ii , iii ) are apparent in the spectra , with feature i decreasing 
 most after exchange . spectra were collected at room temperature over 
 10,000 scans with a delay time of 120 s. using the same methodology , spores of b. subtilis 1a578 were also studied using deuterium  hydrogen exchange 
 via quadrupolar h nmr . figure 2 shows the spectra for spores of b. subtilis 1a578 
 grown in 25% deuterated media before ( a ) and after ( b ) a one - week 
 exchange with h2o , and a comparison of the two spectra 
 on the same axis ( c ) corrected for mass loss during sample preparation . the 1a578 spores appear to have sharper features ii and iii than those 
 of atcc 6051 . likewise , the comparison in figure 2c shows that feature iii is not affected by soaking in h2o whereas features i and ii become smaller . deuterium quadrupolar 
 echo nmr spectra of b. subtilis 1a578 spores : grown 
 in 25% d2o media ( a ) ; same sample 
 after one - week exchange with h2o ( b ) ; a and b shown on 
 the same axis and scaled for mass ( c ) . three main features ( i , ii , 
 iii ) are apparent indicating signals arising from different deuterons 
 in the spore ; feature i exhibits the largest decrease after exchange . spectra were collected at room temperature over 10,000 scans with 
 a delay time of 120 s. spores 
 of both strains of b. subtilis , atcc 6051 and 1a578 , 
 grown in protonated media , were soaked in 99% d2o for 1 
 week and lyophilized , and quadrupolar echo h nmr spectra 
 were collected . the spectrum 
 of wild - type ( atcc 6051 ) spores shows a strong central feature i , 
 an area of intermediate line broadening , and significant broadening 
 at the baseline while features ii and iii are not observed . the chloramphenicol - resistant 
 ( 1a578 ) spores produce a deuterium spectrum that is dominated by feature 
 i and does not have features ii and iii , and broadening of the baseline 
 is present but less intense . deuterium solid - state spectrum of b. 
 subtilis atcc 
 6051 spores grown in protonated medium and soaked in d2o for one week s exchange ( room temperature , 120 s delay , 
 10,000 transients ) . features ii and iii are not apparent , leaving 
 only feature i and a broad signal . deuterium solid - state nmr spectrum of spores of b. subtilis 1a578 ; this strain exhibits a different line shape after h - to - d 
 exchange than b. subtilis atcc 6051 . although the 
 same overall changes ( no features ii or iii and broad signal ) are 
 observed , the broad feature is much less intense . the spectrum was 
 collected at room temperature with a 120 s delay time and a total 
 of 7178 transients . the spectra of bsa lyophilized 
 for 5 days and for 19 days are shown in figures 5b and 5c , respectively . the mobile d2o peak is reduced in the 19-day spectrum versus the 5-day spectrum , 
 with little to no difference in the broader signal . these spectra 
 are identical to previously reported spectra of a deuterated enzyme , 
 subtilisin . however , we previously published 
 a spectrum of deuterated bsa prepared under different conditions . this spectrum is shown in figure 5a and shows a pake doublet with a large separation , similar 
 in width to feature iii in figures 1 and 2 . for this sample , the protein : water ratio ( 500 
 mg:10 ml ) and drying ( 1 day ) were insufficient to achieve complete 
 deuterium exchange and removal of excess water . hence , figure 5a has a lower snr and the additional set of pake 
 doublets are not seen in the samples with extended drying ( figure 5b and 5c ) . a comparison of one sample 
 of deuterated lyophilized bovine serum 
 albumin lyophilized 1 day ( a ) with a second sample ( bsa ) dried for 
 5 days ( b ) and 19 days ( c ) . the pake doublet observed in a is attributed 
 to bound water that vanishes when the sample is dried for longer periods 
 of time ( b , c ) . spectra were collected at room temperature with a 
 delay time of 120 s over 4364 transients ( a ) or 10,000 transients 
 ( b , c ) . figure 6 shows 
 the quadrupolar echo deuterium nmr spectra 
 of the amorphous and crystalline forms of calcium dipicolinate ( cadp ) 
 respectively . the amorphous sample presents itself as having a pake 
 doublet with a peak splitting of 116 khz , with a central peak that 
 is broader than that observed in the b. subtilis spores 
 ( figure 6a ) . when a smaller sample of the amorphous 
 cadp is subjected to additional drying ( figure 6b ) , the pake doublet disappears and the spectrum resembles that of 
 the deuterated bsa samples ( figures 5b and 5c ) . the crystal form exists as a trihydrate , and , 
 when isolated from deuterated water , crystalline cadp3d2o does not show a central peak ( feature i ) , instead revealing 
 a pake doublet with a peak splitting of 31 khz that broadens as it 
 nears the baseline ( figure 6c ) . the crystal 
 structure determined using a small portion of the cadp3d2o nmr sample is shown in figure 7 . h nmr quadrupolar echo spectrum of amorphous deuterated 
 calcium dipicolinate trihydrate dried for 24 h ( a ) , a separate amorphous 
 cadp sample dried for 48 h ( b ) , and crystalline calcium dipicolinate 
 trihydrate ( c ) . a pake doublet surrounds a broad mobile d2o peak in a , which is not evident with longer drying on the other 
 amorphous sample ( b ) ; crystalline cadp shows a pake doublet at 31 
 khz indicative of limited motion ( c ) . spectrum a was collected at 
 room temperature with a delay time of 120 s over 7768 transients ; 
 b over 2200 transients ; c over 10,000 transients . crystal structure of deuterated calcium dipicolinate trihydrate 
 ( cadp3d2o ) , as shown in a thermal ellipsoid plot , 
 showing that it forms a dimer as a crystal . figure 8 shows various spectra collected during the vt nmr 
 experiment involving b. subtilis atcc 6051 . the first 
 spectrum collected ( top left ) is similar to that of figure 1a , with variations caused by lower signal - to - noise 
 ratio in these spectra collected with 2000 scans ( 66 h ) versus 10,000 
 scans ( 333 h ) . when heated to 50 c there are only minor differences 
 observed , mostly a slight decrease in the pake doublet features ii 
 and iii . this change appears to reverse itself when the sample is 
 cooled back to 21 c . feature i becomes dominant and features 
 ii and iii become reduced when the sample is heated to 100 c . as before , the spectrum resumes its previous appearance when cooled 
 back to 21 c ( bottom right ) . spectra obtained for the variable - temperature 
 deuterium nmr of 
 25% deuterated b. subtilis atcc 6051 spores . very 
 slight changes are seen when the spores are heated to 50 c and 
 cooled ; larger changes to the line shape are apparent when heated 
 to 100 c but appear to revert themselves when cooled to the 
 original temperature . these changes and their reversion to previous 
 line shape are suggestive of glass - like transitions inside a rigid 
 spore core . spectra were collected with a delay time of 120 s and 
 2000 transients at the temperatures indicated . the distinct features 
 ( i , iii , and iii ) in the deuterium quadrupolar echo nmr spectrum of 
 partially deuterated b. subtilis atcc 6051 spores 
 ( figure 1a ) are assigned by comparison to samples 
 of known composition . feature i is attributed to deuterons with isotropic 
 motion , possibly from water that is mobile or very dynamic in its 
 environment ; future experiments will examine feature i in more detail 
 to study the t1 of this peak . feature 
 ii has a pake doublet separation ( 32 khz ) similar to that seen in 
 the crystalline calcium dipicolinate sample , whose crystal structure 
 shows water coordinated to the metal ion , carboxylic acids , and nearby 
 water molecules . however , feature ii could also be due to nonlabile 
 deuterons incorporated in biomolecules . using a growth medium with 
 25% d2o thus , methyl groups will be found as ch2d and give a pake pattern with a 38 khz peak separation . the pake pattern is reduced from the typical 
 140220 khz separation found in organic compounds due to free rotation . it is well - known that 
 cd3 molecules in proteins and peptides present 
 a similar nmr spectrum . labile water 
 associated with the lipid bilayers , such as the spore membrane , gives 
 a peak separation of a few kilohertz while deuterons in the lipid 
 itself produce a larger separation , on the order of 30 khz . feature ii could also be interpreted as the result of c2 symmetry 
 jumps ; this is common in crystalline hydrates and , if applicable to calcium dipicolinate trihydrate , would tie 
 feature ii to the spore core as well through the cadp3h2o . attribution of feature iii ( peak separation of 120 
 khz ) to immobilized water molecules has been disputed and was suggested 
 to arise from labile deuterons in biomolecules . we have prepared and studied labile deuterons in the bsa 
 protein sample , and the h nmr spectrum does not have feature 
 iii present after extended drying ( figures 5b and 5c ) . spectra similar to figure 5b have been reported for hydrated subtilisin enzyme 
 isolated from a d2o solution . this sample contains labile 
 o  d and n  d groups along with a layer of hydration water 
 strongly associated with the enzyme . for 
 a sample of bsa dried for 1 day , the spectrum shows a pake doublet 
 with a separation similar to that of feature iii . we attribute the 
 pake doublet in figure 5a to water molecules 
 ( hod ) bound to the protein sample . these hod molecules are not firmly 
 attached to the protein as they can be removed with vacuum drying 
 ( figures 5b and 5c ) . the outer edges of the pake doublet in figure 5a are less sharp than the pake doublet of frozen water ( figure 9c ) . this indicates that excess water associated 
 with bsa is immobilized in a heterogeneous fashion . the variation 
 in chemical environment can be due to localized differences in numbers 
 of neighboring water molecules and the hydrogen bonding network . similar 
 observations are made with cadp samples with a wide pake doublet ( figure 6a ) that is removed with further drying ( figure 6b ) . a comparison of the pake doublets of b. subtilis 1a578 ( a ) versus crystalline cadp3d2o ( b ) , bulk 
 frozen d2o ( c ) , and amorphous cadp3d2o 
 ( d ) , indicating the likelihood of the spore pake doublets arising 
 from immobilized core components . in this model feature iii arises 
 from bound water similar to that of amorphous cadp but unlike bulk 
 water ; feature ii may arise from methyl rotations or from the crystalline 
 cadp3d2o signal . the shape of feature iii ( figures 1 and 2 ) shows that these deuterons are in 
 an environment 
 of substantial homogeneity compared to water associated with amorphous 
 proteins or cadp . if these deuterons are protein - associated water , 
 they would be very susceptible to a temperature - induced increase in 
 isotropic motion . these dynamics should be reversible and would not 
 require the protein itself to undergo isotropic motion . such behavior 
 is shown after heating the spore to 100 c and cooling it back 
 down to 21 c ( figure 8) . the dramatic 
 change of the anisotropic feature iii into the isotropic signal of 
 feature i supports our view that these deuterons are in rigid water 
 associated with biomolecules , and their increased motion does not 
 require a diffuse liquid - like state . within a few degrees of the d2o melting point ( 3.8 c ) used variable temperature h and h nmr studies to examine molecular dynamics in ice . in the 
 hexagonal form of ice , ih , the central water molecule is bound to 2 neighboring h atoms ( via 
 the oxygen ) and 2 neighboring oxygen atoms ( via the protons ) . these 
 four bonds form a tetrahedron around the central water molecule . in 
 wittebort s sample , a narrow line is seen at 6 c 
 and attributed to isotropic reorientation within the ice lattice . 
 at 24 c , our spore samples were analyzed at room temperature , 
 and thus bulk ice is not expected , although the water could be dynamic 
 within a rigid lattice ; the relatively small size of the narrow line 
 at 6 c in wittebort s sample implies that its 
 contribution to feature i would be negligible . the 
 sodium - silicate kanemite has three bound water molecules which move 
 between tetrahedral binding sites within the mineral structure , though 
 some water is found in between layers of the mineral . rigid water in the kanemite sample produces a pake pattern separation 
 > 190 khz due to na  o  d and si  o  d bonds 
 at 120 c . at room temperature , a weak powder pattern is observed symmetric to an isotropic peak , indicating less mobile 
 water undergoing jumps . thus , water in nonbulk environments generates 
 pake patterns with varying quadrupolar coupling constants . report that water in corn starch samples produces pake pattern separations 
 less than 144 khz . thus , it is not unexpected 
 that excess water bound to proteins could give a pake pattern splitting 
 of 120 khz compared to that of 144 khz for bulk ice . dimers 
 of -truxillic acid form with two carboxylic acid 
 groups arranged in a linear fashion . ho = c 
 hydrogen bonds and replacement with deuterium producing a sharp pake 
 doublet with a 117 khz splitting , but 
 only when those carboxylic acids are in the linear arrangement . we 
 do not believe a homogeneous chemical environment such as that found 
 in -truxillic acid , however , produces feature iii in bacterial 
 spores . there are numerous carboxylic acid groups in the spore proteins , 
 but they form hydrogen bonds with amide groups . carboxylic acid groups 
 in the peptidoglycan are arranged in a random disordered fashion and 
 would not be expected to adopt a homogeneous linear conformation . the crystalline calcium dipicolinate unit cell shows one water bond 
 to a carboxylic acid group , yet we do not see feature iii in figure 6c . most of the carboxyl groups are involved in direct 
 chelation of the ca ions , reducing the occurrence of 
 o another 
 possible source of feature iii is nonlabile deuterons found 
 as rigid c  d groups . this labeling would 
 be widespread among the various proteins , dna , peptidoglycan , and 
 membranes found in a mature spore . these deuterons could be responsible 
 for the remaining portion of feature iii at 100 c not converted 
 to the isotropic feature i. thus , we believe that feature iii is due 
 to both nonlabile deuterons in biomolecules and rigid water associated 
 with biomolecules . due to the complexity of the spore sample and the 
 heterogeneous nature of the deuterium spectrum , with many components , we were unable to generate a satisfactory quantitative estimate of 
 each component in the spore . as seen with previous reports , quantitative assignment would be dependent on 
 the level of hydration . nonetheless , our qualitative study provides 
 additional insight in the spore core water environment . future studies 
 will attempt quantification through line shape simulation and comparison 
 of simulated spectra with the spectra reported here . the exchange of accessible 
 deuterons with h2o is demonstrated in figure 1 using spores of b. subtilis atcc 6051 . the 
 decrease in the mobile d2o peak ( feature i ) is expected ; 
 mobile d2o would exchange freely with exogenous water . 
 however , a significant fraction of mobile water is unreachable by 
 external h2o . these deuterons can be both core and noncore 
 water . in the latter case , deuterons are protected from exchange by 
 protein conformations that trap water in a rigid matrix . dormant spores , 
 even hydrated , would maintain proteins in tight conformations which 
 would limit access and exchange as a means of hindering chemical attack . if water can cross the membrane , some core - associated deuterons must 
 be protected from exchange by protein conformations or the transport 
 and exchange of water is very slow compared to the 7-day exchange 
 period . as the exchange was carried out in a water suspension , excess 
 water would cause full hydration and swelling of the spore samples , 
 although in a state of dormancy active water transport would cease 
 to function . a rigid core model would protect water from exchange , 
 but it is also possible that a rigid core could transition into a 
 gel state with excess hydration . if this were the case , some mobile 
 deuterons in protein / dna / cadp environments must be inaccessible to 
 gel water , or the core is a mixture of gel and rigid domains restricting 
 access to exchange sites . nevertheless , this analysis does not preclude 
 the opportunity for gel core water to be present with dynamics different 
 than bulk water . however , experiments 
 targeted to study core - water dynamics after h - to - d exchange would 
 report only accessible water while trapped water would be invisible 
 and not contribute to relaxation rate analysis . from the data in figures 1 and 2 , this trapped water 
 has a noticeable contribution to feature i. thus , kaieda et al . however , it is also possible that the membrane prevents 
 water diffusion . in this scenario , the mobile water peak observed 
 here and in previous work would be due to noncore water molecules 
 protected from exchange by protein conformations . unfortunately , changes 
 to feature i after exchange can not be used to evaluate whether the 
 mobile water peak is due to core or noncore water . it is not unreasonable 
 that the lipid bilayer would undergo significant structural and/or 
 biochemical changes during sporulation to protect the core from dehydration 
 or attack by sporicidal agents through the removal or closure of transmembrane 
 channels and transport proteins , or structural changes to the phospholipids 
 themselves creating a tighter network . deuterium nmr is commonly used 
 to examine the motion of lipid membranes and dynamics within the hydrophobic 
 tails as well as other relevant biomolecules such as dna . if the lipid bilayer deuteron signals could be isolated 
 from other components in the spore , these experiments could be used 
 in the future to elucidate such changes . after lyophilization , the 
 samples are exposed to atmospheric moisture during the 2-week time 
 period required to collect a deuterium nmr spectrum . the rotor , approximately 
 half to three - quarters full , allows for water vapor in the headspace 
 to penetrate the spore and continue exchange processes . we do not 
 detect changes in the spectrum over time or after recollection of 
 prior data with the same sample . drying may cause the 
 spore to acquire hydrophobic properties in the coat protein layers 
 or membrane or change from a gel to rigid core . the inner pake 
 doublet ( feature ii ) also decreases , indicating 
 some of these deuterons are in an environment susceptible to exchange . 
 no change would be seen for nonlabile deuterons in biomolecules created 
 in the culture and sporulation media . if the spore core is impenetrable , 
 water in the calcium dipicolinate trihydrate would remain after exchange 
 and contribute to feature ii , if the core cadp has a structure similar 
 to the crystalline material produced in our laboratory . it is also 
 possible that some of the water associated with cadp is subject to 
 exchange , perhaps on the exterior of crystallites , whereas most of 
 the water in cadp is protected . proposed that these signals could be due to deuterated methyl groups ; 
 similar line shapes were seen in magic - angle - spinning ( mas ) spectra 
 of deuterated valines inside of glycophorin a. interestingly , feature iii is relatively unchanged 
 after soaking 
 in h2o . if this pake doublet were , as suggested , due solely 
 to labile deuterons , the expected result 
 of a deuterium  hydrogen exchange would be a loss of deuterium 
 signal in the spectrum , or at least a decrease in intensity proportional 
 to that seen for features i and ii . as described above , 
 a glassy core would protect labile water deuterons in the core . additionally , 
 if linear hydrogen bonds such as in -truxillic acid dimers are responsible for feature iii , they may persist 
 in water if the biomolecules or peptidoglycan do not rearrange in 
 response to swelling from excess hydration . this seems unlikely as bacillus spores change shape / size in response to external 
 water . if these hydrogen bonds are susceptible 
 to exchange , and responsible for feature iii , then they would be nonexistent 
 in the postexchange spectrum . thus , the persistence of feature iii 
 after the d - to - h exchange process supports either bound water molecules 
 in the spore or the partial deuteration of carbon atoms found in various 
 biomolecules . as described above , we believe that both species are 
 present . hydrogen 
 exchange 
 experiments with a sample of b. subtilis 1a578 spores , 
 as seen in figure 2 . this indicates that changes 
 to features i and ii , and the persistence of feature iii , are not 
 a property confined to the atcc 6051 strain . nevertheless , the spores 
 of the chloramphenicol - resistant and wild - type strains show dramatic 
 differences when subjected to hydrogen - to - deuterium exchange experiments . 
 in these experiments , labile , and accessible , protons were replaced 
 with deuterium by soaking in sterile d2o . the postexchange 
 spectra for atcc 6051 spores show a line shape 
 ( figure 3 ) lacking the sharp features ii and 
 iii of figure 1a . hydrocarbons of biomolecules 
 and lipid groups would not contribute to the post h - to - d exchange 
 spectra . the lack of a strong feature ii reinforces the conclusion 
 that deuterons responsible for features ii and iii are not labile 
 ( perhaps in c  d or chd groups ) or not easily accessible for 
 exchange ( located in the core and/or protected by proteins ) . however , 
 the 1a578 spores produce a spectrum ( figure 4 ) dominated by mobile water ( feature i ) . the broadened baseline has 
 a shape similar to that of the wild - type spores ( figure 3 ) but lower in intensity . proteins external to the core may 
 have a structural conformation that protects labile protons from exchange 
 with deuterons . a proteomics study with the chloramphenicol - resistant 
 1a578 found 85 proteins already identified from bacillus species and 69 novel proteins . thus , 
 unique protein structures may exist that resist exchange with deuterium , 
 a possibility that warrants further study . both spore samples were 
 handled in a similar fashion , thus comparison of the spectra indicates 
 that 1a578 has a greater fraction of mobile water . this could result 
 from the chloramphenicol - resistant spores retaining a greater mass 
 of mobile water , having a mobile water fraction with greater resistance 
 to drying , or the labile deuterons being predominately found as mobile 
 water . these deuterons obviously have rapid isotropic motion , but 
 it is not clear if they are located in the core , noncore regions , 
 or both . if feature iii of the spore deuterium nmr spectrum 
 in figure 1a was indicative of labile deuterons , 
 asserted by 
 kaieda et al . , the spectrum observed 
 in figure 3 should look identical ( or nearly 
 so ) to that seen in figure 1a . instead , figure 3 shows a feature ii that is practically indistinguishable 
 from the central mobile deuterium peak and broad shoulders instead 
 of the sharp pake doublet seen in feature iii of figure 1a . do = c 
 hydrogen bonds susceptible to exchange , the h - to - d experiment should 
 produce a sharp pake doublet . instead , we speculate that the broad 
 signals in figures 3 and 4 are from labile deuterons of proteins . when the spectra in figures 3 and 4 are compared to a 
 spectrum of bsa , the result is a nearly identical overlap for the 
 wild - type 6051 spores ( figure 10 ) . however , 
 with its large fraction of mobile water , the resistant 1a578 spores 
 ( figure 11 ) do not overlap with the bsa spectrum 
 unless the protein spectrum is reduced in height . while the scaling 
 factor was 3.3 , this number is somewhat meaningless as we are not 
 convinced that labile deuterons in the coat proteins of 1a578 spores 
 would be exchanged to a similar degree as labile deuterons in a homogeneous 
 solution of bsa . postexchange b. subtilis atcc 6051 spore 
 spectrum 
 ( figure 3 ) to which the five - day - dried bsa 
 spectrum ( figure 5b ) has been overlaid . postexchange b. subtilis 1a578 spore spectrum 
 ( figure 4 ) overlaid on the same spectrum as 
 the five - day lyophilized deuterated bsa ( figure 5b ) . bsa s spectrum fits well to the broad peak feature in 
 1a578 but less so to the mobile water peak feature . the difference 
 in line shape between this and figure 10 is 
 attributed to structural changes in b. subtilis 1a578 
 resulting in higher protein conformational homogeneity and may be 
 responsible for the differences in line shape overall between b. subtilis 1a578 and b. subtilis atcc 
 6051 . we speculate that the spore core is more a solid than 
 a gel - like state , but exchange experiments alone do not provide enough 
 evidence to distinguish the physical state of the core . instead , we 
 base our analysis on variable - temperature data collected to provide 
 this distinction . figure 8 shows the results 
 of the vt nmr experiments with b. subtilis atcc 6051 
 spores . it is evident that the mobile d2o peak does not 
 increase perceptibly when the sample is heated from 21 to 50 c . however , when cooled back to 21 c and heated to 100 c , 
 the central peak intensity increases dramatically . the high temperature 
 causes significant portions of the spore to undergo isotropic , or 
 nearly isotropic , motion . other portions of the spore retain anisotropic 
 behavior as evidenced by the persistence of features ii and iii after 
 heating . the lack of a dramatic change in features ii and iii at 50 
 c suggests that components able to undergo isotropic motion 
 are not affected by the moderate heating . this analysis does not support 
 a gel - like core with a high mobile water content , which would be expected 
 to show gradual changes as the temperature is increased . the rigid 
 core analysis is similar to the predictions made by sapru indicating glass - transition temperatures in 
 between 50 and 100 c for various bacillus strains . the analysis of our vt nmr data builds upon our assignment of spectra 
 to different components , drying effects on the spectra , and changes 
 caused by h / d or d / h exchange . as discussed below , we suggest a spore 
 model with a rigid core where cadp and biomolecules are rigid ( features 
 ii and iii ) with a smaller fraction of deuterons existing as nonrigid , 
 perhaps mobile , water ( feature i ) . data collected at 100 c suggests 
 that there is a phase change between 50 and 100 c responsible 
 for the large increase in the isotropic deuteron signal . the higher 
 fraction of mobile deuterons could arise from faster dynamics of nonlabile 
 deuterons in biomolecules . nevertheless , 100 c is insufficient 
 to convert every deuteron of features ii and iii into an isotropic 
 state . deuterated peptidoglycan , contributing to feature iii , is a 
 highly cross - linked network and not expected to have large degrees 
 of freedom , even with heating . also , these deuterons may exist in 
 proteins that resist conformation changes at high temperature . crystalline 
 cadp may generate feature ii , and work in our lab shows that crystalline 
 cadp does not melt in boiling water . when the sample is cooled 
 back to 21 c ( from 100 c ) , 
 nmr data show that the sample characteristics which generate the room 
 temperature spectra are nearly identical to those of the initial spectra 
 collected at 21 c prior to heating . deuterated 
 biomolecules would return to an immobile state , but we do not know 
 if these biomolecules have been denatured to render the spore inert . previous work by the setlow lab has shown that the viability of b. subtilis spores is relatively unaffected by dry heating 
 to 100 c . the viability of these 
 spores also suggests that the protein denaturation is modest and would 
 not generate the isotropic signal at 100 c . thus , changes to 
 feature iii in figure 8 are attributed to bound 
 water molecules where heating increases their motion to generate an 
 isotropic peak , yet cooling allows them to revert to the bound state 
 without affecting the viability of the spore itself . from this viewpoint , the vt nmr data suggest the presence of bound water contributing to 
 feature iii in contrast to the assertions promoted by kaieda et al . proton nmr has been used to examine water in spores ; however , the 
 combination of heterogeneous samples and narrow chemical shift dispersion 
 creates difficulties in assigning spectra . nevertheless , the water 
 will generate a very strong signal , and its chemical environment will 
 influence spin relaxation rates . bradbury et al . reported equal amounts 
 of water in core and cortex + coat regions . water was found to be 
 both mobile and immobile . after separating the coat proteins and pg 
 cortex , the water in these samples was found to be immobile ; thus , 
 mobile water was assigned to the core . using solid - state nmr spectroscopy , the characteristic c resonance for dipicolinic acid ( dpa ) was detected with cross - polarization in freeze - dried and rehydrated 
 spores of b. subtilis . cpmas data reflect those c spins that maintain strong 
 dipolar coupling with nearby h nuclei , and thus indicate 
 limited or absent molecular motion . in contrast , single - pulse mas 
 ( spmas ) signals are strongly affected by mobile species where strong 
 spin  lattice effects cause fast t1 relaxation . leuschner and lillford could not detect dpa with c spmas , demonstrating that the dpa is in a firm rigid lattice , 
 and rehydration of the core does not increase the molecular motion 
 of dpa . from these prior studies , the core - specific dpa molecules 
 may exist in a glassy core with water around the dpa also in an immobile 
 state . we have shown in figure 9b that the 
 trihydrate form of crystalline cadp produces a deuterium spectrum 
 with a pake doublet similar to feature ii . this does not prove that 
 cadp in the core is crystalline , only that it is possible that cadp 
 could adopt a structural arrangement similar to the crystal structure . it is entirely possible that the cadp is found as microcrystals in 
 a glassy matrix of water , dna , and proteins . the d - to - h exchange experiments 
 show slight reduction of feature ii ; this reduction could be due to 
 exchangeable waters around the cadp crystallites . reported a quadrupolar 
 echo h nmr spectrum of fully hydrated mn depleted b. subtilis ps533 spores and concluded that the broad 130 
 khz feature was entirely the result of labile deuterons in immobilized 
 proteins and other biomolecules in the spore . could state that there was no 
 evidence for immobilized water in the core if their data and experimental 
 techniques could not detect immobile core - associated d2o . their protocol is essentially a hydrogen - to - deuterium exchange 
 experiment introducing d2o after growing spores in deuterium - free 
 media ; however , instead of lyophilization , the authors used the wet 
 spore pellet for their nmr analysis . the line shape observed in figures 10 and 11 ( particularly figure 11 ) is similar to that shown in kaieda et al . for their quadrupolar echo h nmr 
 results . in their paper the authors claim that the wet pellet spectrum demonstrates 
 swollen spores with a gel - like core and that their relaxation rate 
 measurements reflect the dynamics of water within the core region . we do not dispute the validity of their data collection or their observation 
 of water fractions with bulk and nonbulk behavior . we assert that 
 their measurements are not representative of all water molecules in 
 the spore sample . the persistence of feature i after d - to - h exchange 
 suggests that water remains trapped , perhaps in a rigid core as suggested 
 by our vt nmr data . samples of 1a578 spores generated from protonated 
 media and followed by deuterium exchange produce a spectrum ( figure 11 ) similar to that in kaieda s work . we believe 
 that our spectrum reflects the mobile water content ( core and noncore ) 
 of a dried spore sample while kaieda s data originates with 
 fully hydrated samples . upon closer inspection , their spectrum shows 
 a broadened baseline similar to that seen with bsa protein reported 
 here , and we agree that this baseline broadening is due to labile 
 deuterons in biomolecules . lastly , sunde et al . assert that , were the core in a glassy state , there would 
 be a broad signal apparent on a h nmr spectrum of the 
 spore ; however , such a signal would not be detectable using the magnetic 
 relaxation dispersion experiments as performed . we have observed this 
 signal in different spore strains , and its width of 120 khz 
 is consistent with excess water bound to bsa and cadp . are similar to that in figure 11 ( the b. subtilis 1a578 ) , and why there are 
 differences in the line shape between b. subtilis atcc 6051 and 1a578 , may have an explanation . while atcc 6051 is 
 a wild - type strain , it is possible that the mechanism of resistance causes structural 
 changes in the vegetative cells of this strain . such changes might 
 manifest themselves in subtle alterations in the spore structure resulting 
 in the differences in the deuterium nmr spectra of figures 14 . the strain used 
 both in kaieda et al.s work and 
 in the paper by sunde et al . thus , the similarity of the deuterium nmr spectra 
 reported by kaieda et al . to b. subtilis 1a578 spectra 
 shown in figure 4 may result from introducing 
 a resistance locus to b. subtilis . from this viewpoint , it is also noteworthy that features ii and 
 iii of the wild - type b. subtilis atcc 6051 spores 
 ( figure 1 ) show greater broadening and heterogeneity 
 compared with the sharper lines seen in figure 2 , data from chloramphenicol resistant strain . sharper lines result 
 from enhanced structural homogeneity , and thus the resistance locus 
 appears to have caused an observable reduction in variation of proteins / biomolecule 
 conformation . this observation merits additional study with other 
 bacterial spore strains . it may be possible to apply other sophisticated 
 biomolecular nmr experiments to explore structural biology via c and n labeling in 
 this work , we studied deuterium nmr spectra of b. subtilis atcc 6051 and 1a578 with an aim of determining the state of water 
 inside of the spore core . this was aided by variable - temperature nmr 
 studies of these species and h nmr spectra of calcium 
 dipicolinate and bovine serum albumin . the overall nmr line shape 
 of spectra for spores of both b. subtilis strains 
 indicates that water is in multiple distinct states in the spore . 
 the wide pake doublet and the maintenance of its signal through deuterium hydrogen 
 exchange indicates that it is bound water , likely associated with 
 the core . deuterium 
 exchange spectra where the only signals that appear on the spectrum 
 are those similar to bulk proteins such as those seen in deuterated 
 bovine serum albumin . the difference between the spore spectrum shown 
 in kaieda et al . and that of b. subtilis atcc 6051 is postulated to be due to structural 
 differences perhaps attributable to different proteins or antibiotic 
 resistances , similar to b. the crystalline 
 calcium dipicolinate spectrum , when compared to spore spectra , suggests 
 that the inner pake doublet may be attributable to immobile core components . in addition , the lack of proportional increase in the mobile deuterium 
 signal during the variable - temperature nmr experiments , and the return 
 of the signal to its original line shape upon cooling , suggest a narrow 
 transition along the lines predicted by sapru and ablett and thus a glassy core . the existence of bound water has come under recent scrutiny from the measurement of deuterium relaxation 
 rates . these samples were dispersed in deuterated water , and deuterium 
 relaxation rates were measured with solution - state nmr methods at 
 different magnetic field strengths . lattice relaxation 
 could be explained by molecular motions on the order of 50 ps , which 
 implies a mobile water fraction within the core . as described by the 
 authors , this correlation time is too fast for water to be in a  rigid 
 lattice  environment that would produce a 200 khz wide spectrum . as shown in this work , deuterium wide line nmr 
 spectroscopy is able to detect both mobile and rigid lattice waters 
 simultaneously . our data demonstrate the existence of rigid lattice 
 water molecules in bacterial spores and that these molecules are in 
 a different chemical environment than water molecules within the tetrahedral 
 bonding network of frozen water . additional data show that immobilized 
 water in spores is resilient and unable to undergo chemical exchange 
 with water molecules in exogenous solution .
dormant bacterial spores are able 
 to survive long periods of time 
 without nutrients , withstand harsh environmental conditions , and germinate 
 into metabolically active bacteria when conditions are favorable . 
 numerous factors influence this hardiness , including the spore structure 
 and the presence of compounds to protect dna from damage . 
 it is known 
 that the water content of the spore core plays a role in resistance 
 to degradation , but the exact state of water inside the core is a 
 subject of discussion . 
 two main theories present themselves : either 
 the water in the spore core is mostly immobile and the core and its 
 components are in a glassy state , or the core is a gel with mobile 
 water around components which themselves have limited mobility . using 
 deuterium solid - state nmr experiments , we examine the nature of the 
 water in the spore core . 
 our data show the presence of unbound water , 
 bound water , and deuterated biomolecules that also contain labile 
 deuterons . deuterium 
 hydrogen exchange experiments show that 
 most of these deuterons are inaccessible by external water . 
 we believe 
 that these unreachable deuterons are in a chemical bonding state that 
 prevents exchange . 
 variable - temperature nmr results suggest that the 
 spore core is more rigid than would be expected for a gel - like state 
 . 
 however , our rigid core interpretation may only apply to dried spores 
 whereas a gel core may exist in aqueous suspension . nonetheless , the 
 gel core , if present , is inaccessible to external water .