text:
Isolation of Nitrospira belonging to Sublineage II from a Wastewater Treatment Plant

Nitrite oxidation is a key step in nitrogen removal in biological wastewater treatment plants. Recently, two phylogenetically different Nitrospira (sublineages I and II) have been recognized as the numerically dominant nitrite-oxidizing bacteria in wastewater treatment plants. However, Nitrospira sublineage II inhabiting activated sludge was not isolated and its detailed properties were unclear. In this study, we developed a new method for the isolation of Nitrospira forming micro-colonies using a cell sorter. We obtained a novel pure strain “Nitrospira japonica” from the activated sludge. Subsequently, phylogenetic and physiological analyses revealed that Nitrospira japonica belongs to sublineage II and grew in medium containing formate. This method has the potential to isolate other uncultured microorganisms forming micro-colonies.

Nitrite oxidation is an important step in nitrogen removal in biological wastewater treatment plants. Nitrite-oxidizing bacteria (NOB) involved in nitrite oxidation in wastewater treatment plants are known to belong to the genus Nitrobacter or the genus Nitrospira. Previously, the physiological and biochemical properties of NOB were mainly investigated using Nitrobacter strains because they were easily cultured in the laboratory. However, cultivation-independent molecular methods (5,10,11,27) and immunological techniques (3) indicated that members of the genus Nitrospira and not Nitrobacter species were dominant NOB in aquaria and fullscale wastewater treatment plants.
The comparative analysis of 16S rRNA gene sequences revealed that the genus Nitrospira was classified into four different phylogenetic lineages, and sublineages I and II play a key role as nitrite oxidizers in wastewater treatment plants (6). Because Nitrospira-like bacteria in nitrifying activated sludge or biofilms are fastidious and slow-growing organisms, their physiological properties have been investigated by molecular-based approaches (6,18,21,28). However, high enrichment or pure culture is required to determine detailed biochemical properties and genomic information. Recently, "Candidatus Nitrospira defluvii" belonging to sublineage I was enriched from activated sludge, and its physiological properties were characterized (23). Moreover, the complete genome of "Ca. Nitrospira defluvii" allowed insight into the key metabolic pathways of Nitrospira and its evolution (14). Meanwhile, no Nitrospira within sublineage II has been isolated from wastewater treatment plants so far.
The major members of the genus Nitrospira sublineage II in activated sludge are known to be phylogenetically distant from Nitrospira moscoviensis, which was isolated from an urban heating system in Moscow and classified as sublineage II (7).
Here, we isolated pure cultures belonging to sublineage II from activated sludge. Previously, we enriched sublineage II Nitrospira from activated sludge using a continuous feeding bioreactor (9), but could not obtain pure cultures partly because growing Nitrospira cells tended to form microcolonies. In this study, we developed a new isolation method, which enabled selective separation of Nitrospira microcolonies from the enrichment culture via a cell-sorting system. As a result, we achieved pure culture of sublineage II Nitrospira from activated sludge.

Source of bacteria
We enriched Nitrospira-like bacteria using a continuous feeding bioreactor (9). The inoculum was obtained from the nitrification stage of the municipal wastewater treatment plant in Ochiai, Tokyo, Japan, in February 2009. For the following cell-sorting experiments, we used the enrichment sample taken from the bioreactor where the ratio of Nitrospira sublineage II to the total microorganisms was 33.6%.

Sorting of micro-colony
We selectively separated Nitrospira micro-colonies from the enrichment samples using a cell-sorting system (FACS Aria; BD Biosciences, Franklin Lakes, NJ, USA). The samples were dispersed by ultrasonic treatment (Sonifier II model 150; Branson, Danbury, CT, USA) for 1 min, and were filtered successively through filter papers (pore size 35 µm, BD) to remove large cell aggregates. Filtered samples were then applied to a cell sorter, with the sample flow rate adjusted to approximately 100-300 events per second in single cell mode. The dot plot area defined on a two-parameter histogram (forward scatter [FSC] versus side scatter [SSC]) was identified. At least 10,000 particles were analyzed for each histogram. Fractions separated from each region (P1-P6) of the dot plot area were mounted onto glass slides (at least 500 particles per slide) and investigated by fluorescence in situ hybridization (FISH) analysis. In each fraction, the ratio of Nitrospira to total microbial cells was calculated by direct counting. Subsequently, a fraction containing mostly Nitrospira micro-colonies was identified. Samples within this fraction were sorted and inoculated into 96-well microtiter plates for sub-cultivation.

Chemical analyses
Nitrite-nitrogen and nitrate-nitrogen concentrations were determined quantitatively by ion chromatography (IC 2001; Tosoh, Tokyo, Japan). Influent/effluent samples in the bioreactor and nitrite oxidation activity test samples were filtered through 0.2 µm cellulose acetate membrane filters (Advantec, Tokyo, Japan).

DNA extraction and polymerase chain reaction (PCR) amplification
DNA was extracted from primary enrichment samples using the ISOIL extraction kit (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The 16S rRNA gene fragment (ca. 1,500 bp) of the total DNA was amplified using primers, 27f: 5'-AGAGTTTGATCATGGCT-3' and 1492r: 5'-TACGGTTACCT TGTTACGACTT-3'. The following thermal profiles were used in 16S rRNA gene amplification: an initial denaturing step was conducted at 95°C for 2 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 1 min. The final extension step was conducted at 72°C for 2 min. The PCR reaction mixtures (50 µL) contained 10× PCR buffer, 20 µM of each primer, 2.5 mM dNTPs, and Ex Taq DNA polymerase (Takara Bio, Shiga, Japan).

Phylogenetic analysis
Amplified PCR products were purified using the Wizard SV Gel and PCR Clean up System (Promega, Tokyo, Japan). Purified PCR products were sequenced using primers, 27f: 5'-AGAGTTTGAT CATGGCT-3' and 1492r: 5'-TACGGTTACCTTGTTACGACTT-3', by Fasmac (Kanagawa, Japan). Finally, the 16S rRNA gene sequences, comprising approximately 1,500 bases, were determined. Alignment editing and phylogenetic analyses were performed using ChromasPro version 1.4.1 software (Technelysium Pty, Tewantin, Australia) and MEGA4.0 software (25). The bacterial 16S rRNA gene sequences were compared with those available from the DNA Data Bank of Japan (DDBJ) database. Genetic distance was calculated using a p-distance model of nucleic acid substitution.

Electron microscopy
Ultrathin sections of isolate were prepared and observed under transmission electron microscopy (TEM). Isolate cells were fixed in 2% glutaraldehyde in 0.1M phosphate buffer, and post-fixed in 2% osmium tetroxide for 2 h at 4°C. The specimens were dehydrated in a graded series of ethanol, placed in propylene oxide, and embedded in epoxy resin (EPON812) for 48 h at 60°C. Ultrathin sections were cut and stained with uranyl acetate and lead citrate prior to examination under TEM (JEM-1200EX; JEOL, Tokyo, Japan) at 80 kV. Images of the isolate were obtained under a scanning electron microscope (SEM). Following fixation and dehydration, specimens were placed in isoamyl acetate, critical point dried, coated using an osmium plasma coater and examined under a SEM (JSM-6320F, JEOL) at 5 kV.

Physiological analyses Nitrite oxidation activity test
Nitrite oxidation activity tests for optimum nitrite concentration were performed at 23°C in 50 mL test tubes filled with 10 mL mineral nitrite medium (5-75 mg-N L −1 ). Media were stirred to ensure sufficient oxygen supply. Optimal temperature for nitrite oxidation was investigated within the range 10-46°C in test tubes filled with 10 mL mineral nitrite medium (20 mg-N L −1 ).

Growth activity test
We investigated the growth of Nitrospira cells in nitrite medium or organic medium. All incubations were performed in 300 mL Erlenmeyer flasks containing 100 mL of nitrite medium or organic medium at 30°C under dark conditions. The pH was adjusted to 7.5-7.8. Media were stirred to ensure sufficient oxygen supply. The composition of the nitrite medium was the same as described in pure culture. The organic medium comprised one of five types of organic matter (550 mg L −1 sodium pyruvate, 1.

Sorting of micro-colony
Previously, we enriched Nitrospira-like bacteria belonging to sublineage II from a wastewater treatment plant using a continuous feeding bioreactor (9). Nitrospira-like bacteria belonging to sublineage II had been enriched in the bioreactor for over two years. FISH direct counting analysis revealed that the rate of Nitrospira in sublineage II to the total microorganisms in the enrichment sample was 33.6%. Additionally, observations by FISH analysis indicated that enriched Nitrospira cells tended to form into densely packed spherical micro-colonies, which contained only Nitrospira sublineage II cells (Fig. 1A). Meanwhile, most planktonic cells were other microorganisms except Nitospira in sublineage II and uneven-shaped aggregates were constructed by multiple types of microorganisms.
Thus, we conceived that the cell-sorting system could separate Nitrospira belonging to sublineage II from the enrichment sample by exploiting the shape of micro-colonies. The cell-sorting system enables the sorting of micro-particles based on their light-scattering or fluorescent properties. In this experiment, forward scatter (FSC) and side scatter (SSC) were used for particle sorting. In principle, the magnitude of FSC and SSC signals reflects the size of the particles and their complexity, respectively. Thus, micro-colonies exhibited relatively high FSC and low SSC signals because they were large and densely spherical. Meanwhile, most planktonic cells exhibited low FSC and uneven-shaped aggregates exhibited high levels of both signals because of their size and rough surface. Applying the concept described above, Nitrospira micro-colonies were physically separated from the particle mixture by their distinctive light-scattering signature using the cell-sorting system without specific labeling.
Initially, the enrichment sample taken from the bioreactor was sonicated and applied to the cell sorter, and the dot plot area was identified (Fig. 2). Six sub-areas (P1-P6) were defined in the dot plot area, and 100 particles separated from each area were mounted onto glass slides and observed under a fluorescent microscope. As expected from the above hypothesis, FISH analysis confirmed that P1 and P2 areas (low FSC fraction) included most planktonic cells, the P3 area (high FSC and high SSC fraction) included larger and more complex multi-species aggregates, P4-P6 areas (high FSC and low SSC fraction) included many pure Nitrospira micro-colonies, and the P6 area in particular included the most pure Nitrospira micro-colonies. Indeed, FISH observations of the P6 area revealed that 43 out of 100 particles mounted onto glass slides were pure Nitrospira microcolonies (Fig. 1B).

Isolation and subculture
Single micro-colonies (Nitrospira micro-colonies) collected from the P6 area were individually inoculated into 96well microtiter plates containing nitrite medium (10 mg-N L −1 ). In total, 252 wells were used for subculturing tests. After two months of incubation under dark and static conditions, the growth of Nitrospira sublineage II was confirmed in 4 out of 252 wells under microscopic observation by FISH analysis. The 4 strains were identified as J1-J4 strains. FISH analysis confirmed the growth of other species, the growth of multiple species, or no growth in remaining wells (248 wells). The J1-J4 strains were transferred into 5 mL vials containing 1 mL fresh nitrite medium. The pure cultures were then up-scaled to 100 mL plastic tubes containing 20 mL fresh nitrite medium, and finally to 2 L Erlenmeyer flasks containing 500 mL fresh nitrite medium. The purity of the 4 strains was checked by FISH analysis, DGGE analysis, spread-plating onto Luria-Bertani solid medium and R2A solid medium, and inoculating diluted Nutrient Broth liquid medium (Supplementary materials). As a result, the J1-J4 strains were confirmed as absolutely pure cultures. Although micro-colonies of Nitrospira pure culture consisted of 10-100 cells, DGGE analysis demonstrated that only one band was obtained. Thus, all cells of Nitrospira micro-colonies were identical clones.

Phylogenetic analysis
Phylogenetic analysis of 16S rRNA genes revealed that the J1-J4 strains were highly similar to each other (99.5-100% sequence similarity). Thus, these four strains were one species, and we identified J1 strain as a representative strain "Nitrospira japonica". Comparative analysis of 16S rRNA gene sequence of Nitrospira japonica with that of other Nitrospira strains revealed that Nitrospira japonica belonged to sublineage II (6), but was distantly related to Nitrospira moscoviensis, the only described species in sublineage II with 95.6% similarity (7). The 16S rRNA gene sequence of Nitrospira japonica shared a relatively high level of identity with the sequences of clones OWP-10 and OWP-21 from enrichment samples in a previous study (97.9 and 98.1%, respectively) (9), and shared the highest level of identity with the sequence of Uncultured Nitrospira sp. clone S1-62 (HQ674926 [98.8%]), which was obtained from weathered feldspar mineral. The phylogenetic tree was constructed on the basis of 16S rRNA gene sequences of selected Nitrospira isolates and environmental clones (Fig. 3). Additionally, clones obtained previously from soils and activated sludge were respectively classified into separate clusters. Although Nitrospira japonica was isolated from activated sludge, its 16S rRNA gene sequence shared a relatively higher level of identity with the sequences of soil cluster compared with the activated sludge cluster.

Morphology
Micro-colonies of Nitrospira japonica consisted of 10-100 cells and the average size of micro-colonies was 5-20 µm (Fig. 4A). SEM observation revealed that Nitrospira japonica cells were densely packed in a micro-colony (Fig. 4B). Meanwhile, planktonic Nitrospira japonica cells ranged from 0.3 to 0.5 µm in width and from 0.5 to 0.7 µm in length, and the cell was a curved rod shape. TEM observations of ultrathin sections of Nitrospira japonica revealed that the cells were embedded in extracellular polymeric substance (EPS) (Fig.  4C). Previously, it was reported that the shape and arrangement of "Ca. Nitrospira defluvii", which was enriched from activated sludge, changed with nitrate accumulation and increasing cell density after prolonged incubation (23). Likewise, micro-colonies of Nitrospira japonica changed to planktonic cells after prolonged incubation in this study.

Nitrite oxidation activity test
Nitrospira japonica grew aerobically as a chemolithoautotrophic nitrite oxidizer in inorganic medium containing nitrite as the sole energy source and bicarbonate as the sole carbon source. Nitrospira japonica consumed nitrite with equivalent production of nitrate. By incubating the strain under different nitrite concentrations (5-75 mg-N L −1 ), the optimal nitrite concentration for growth of Nitrospira japonica was determined as 20 mg-N L −1 (Fig. 5A). Moreover, at this nitrite concentration, the temperature dependence of Nitrospira japonica growth was examined across the temperature range 10-46°C. Nitrite-oxidizing activity was observed between 22-34°C inclusive, but there was no observation of nitrite oxidation at 10 and 46°C (Fig.  5B). As a result, the optimal temperature was 31°C.

Growth activity test
Growth activities of Nitrospira japonica were investigated in nitrite medium and in organic medium, which contained no nitrite and one of the five types of organic matter (pyruvate, glucose, peptone, acetate, and formate). At culture start-up, 5 mL pure culture was supplied with 95 mL of each medium. Initially, Nitrospira cells were not detectable under microscopic observation. After 20 days of incubation in nitrite medium, growth of Nitrospira japonica was confirmed by microscopic observation after DNA staining. After 40 days of incubation, growth of Nitrospira japonica was confirmed in medium containing no nitrite or formate. However, Nitrospira japonica did not grow in medium containing other organic substrates.

Discussion
In this study, we successfully isolated a novel strain "Nitrospira japonica" from an enrichment culture, which was originally enriched with activated sludge from a wastewater treatment plant (9). Nitrospira japonica is the second isolate belonging to sublineage II of the genus Nitrospira. Previously, Nitrospira moscoviensis was obtained as a pure culture belonging to sublineage II from a Moscow heating system (7). However, sublineage II contains 16S rRNA sequences of uncultivated bacteria retrieved from diverse habitats, including bioreactors, freshwater aquaria, and soil (1,6,8,10), which implies that the physiological properties of Nitrospira strains belonging to sublineage II could be different from those of Nitrospira moscoviensis. Thus, it is worth that a second Nitrospira pure culture belonging to sublineage II was obtained from activated sludge and its physiological properties were investigated. Comparison of the physiological properties of Nitrospira japonica with those of other Nitrospira strains indicated that Nitrospira japonica was more similar to "Ca. Nitrospira defluvii" than Nitrospira moscoviensis in some respects (tendency to aggregate, optimal temperature, and utilization of organic substrate) ( Table 1). Comparative analysis of the 16S rRNA gene sequence showed that Nitrospira japonica had higher similarity to Nitrospira moscoviensis than "Ca. Nitrospira defluvii" (95.6 and 92.7%, respectively), suggesting a difficulty in linking the phylogenetic similarity to physiological properties.

Growth ability of micro-colony
We successfully isolated Nitrospira japonica from an enrichment culture by selective inoculation of Nitrospira micro-colonies via a cell-sorting system. One hundred particles within the P6 sub-area mounted onto glass slides were included 43 pure Nitrospira micro-colonies, and the total number of wells into which Nitrospira micro-colonies had been inoculated was estimated as 109 out of 252 wells (43%). However, the number of wells in which Nitrospira growth was confirmed during two months of incubation was only 4. In other words, the growth ability of Nitrospira microcolonies was 3.67% (4 out of 109 wells). This growth ability was possibly low because Nitrospira micro-colonies did not grow due to damage from the sorting method (sonication and Fig. 3. Phylogenetic analysis showed the affiliation of isolates in this study. The phylogenetic tree was based on 16S rRNA gene sequences of selected Nitrospira. The tree was constructed using the neighbor-joining algorithm. Numbers at branch nodes are bootstrap values. Previously defined sublineages (6) of the genus Nitrospira (Roman numbers) are shown. Clones obtained previously from soils and activated sludge are classified into separate clusters. Scale bar corresponds to 1% estimated sequence divergence.
sheath fluid of the cell sorter) or Nitrospira micro-colonies did not still grow to a detectable level into 96-well microtiter plates under a fluorescent microscope.

Use of organic substrates
Previously, it was reported that "Ca. Nitrospira defluvii" and Nitrospira marina benefit from simple organic compounds in nitrite media (23,29), and uncultured Nitrospira in sewage plants take up pyruvate (6). Additionally, it was discovered that the genome of "Ca. Nitrospira defluvii" encodes pathways for the catabolic degradation and assimilation of several organic substrates (14). Meanwhile, the (A) In situ hybridization was performed with Cy3-labeled probe Ntspa1151, specific for Nitrospira sublineage II (red) and mixed FITClabeled probes of EUB338, EUB338II, and EUB338III, for the detection of all bacteria (green). Nitrospira japonica appeared yellow due to binding with both Cy3-labeled and FITC-labeled probes. Scale bar is 5 µm.  chemo-organotrophic growth of other pure strains belonging to the genus Nitrospira was not observed (7,12,13). Thus, members belonging to the genus Nitrospira were physiologically distinguished as two types, mixotrophic Nitrospira and obligately chemolithoautotrophic Nitrospira. We assumed that Nitrospira japonica isolated in this study was also a mixotrophic type.
Previously, it was difficult to isolate pure strains which have the ability to form micro-colonies like Nitrospira in activated sludge by dilution series or plating techniques (24). However, the present work describes the successful isolation of Nitrospira by taking advantage of micro-colonies. If targeted microorganisms form micro-colonies, they can be isolated using our developed method. We could apply this new method to the isolation of uncultured microorganisms, which can be separated based on the properties of their microcolonies via a cell-sorting system.


extfieldsofstudy:
Materials Science
Medicine

s2fieldsofstudy: Biology Physics

text:
Rapid internal contraction boosts DNA friction

Macroscopic objects are usually manipulated by force and observed with light. On the nanoscale, however, this is often done oppositely: individual macromolecules are manipulated by light and monitored with force. This procedure, which is the basis of single-molecule force spectroscopy, has led to much of our quantitative understanding of how DNA works, and is now routinely applied to explore molecular structure and interactions, DNA–protein reactions and protein folding. Here we develop the technique further by introducing a dynamic force spectroscopy set-up for a non-invasive inspection of the tension dynamics in a taut strand of DNA. The internal contraction after a sudden release of the molecule is shown to give rise to a drastically enhanced viscous friction, as revealed by the slow relaxation of an attached colloidal tracer. Our systematic theory explains the data quantitatively and provides a powerful tool for the rational design of new dynamic force spectroscopy assays.

L ife literally is a tour de force. And much of the mechanical load exerted on tissues and cells is supported and transmitted by biomolecules that can be classified as semiflexible polymers 1 . Already at physiological force levels, semiflexible biopolymers exhibit non-Hookean stretching behaviour, which sets them apart from other nanoscopic forcebearing elements, such as AFM tips or optical tweezers. Their characteristic force-extension relation under static tension [2][3][4] has become the workhorse of a whole industry of single-molecule force spectroscopy methods, employing the polymers as gauges, linkers 5 , molecular handles 6 , or as a virtual magnifying glass to explore the molecular world at ever higher spatial 7,8 and temporal 9 resolution. However, the mechanical nonlinearity substantially complicates the dynamic response to external perturbations. If you excite an overdamped linear spring, the excitation always decays exponentially and the spring attains its equilibrium state within a characteristic relaxation time. In contrast, polymeric response curves exhibit no well-defined relaxation timescale and their relaxation generally passes through a multitude of power-law regimes, subtly dependent on the details of the applied force protocol 10,11 . Mathematically speaking, the response is not governed by the relaxation of a single eigenmode (a single equivalent spring-dashpot element) but by a whole hierarchy of bending fluctuations of different wavelengths and their corresponding relaxation times. As demonstrated below, such complex mode superpositions can give rise to somewhat unintuitive and sometimes surprising dynamical effects.
A major source of complication in dynamics, compared with the better understood stationary situation, lies within the intricate coupling of the conformational dynamics of a stretched polymer to the solvent hydrodynamics. In its equilibrium conformation, as a coil of radius R, the polymer has a friction coefficient comparable to that of a colloidal bead of about the same radius. In contrast, if the polymer has a straight, rod-like conformation, it feels a friction force proportional to its contour length L4 4R. For a l-phage DNA, L/RE20, which underscores how important the effect can become. The result of a more quantitative theoretical analysis for our specific experimental set-up is illustrated in Fig. 1. It shows that the friction coefficient of a DNA-coated colloidal bead (Fig. 1b) would increase by E30% if one of the molecules of its coating layer could be permanently transformed into a straight conformation of length L ¼ 16 mm (Fig. 1c). The purpose of the following discussion is to demonstrate both experimentally and theoretically that a much more dramatic, roughly fourfold, friction enhancement can be achieved if the initially stretched polymer is additionally allowed to recoil dynamically (Fig. 1d). At the origin of this observation lies an excess viscous friction that is self-generated by the internal conformational dynamics. To our knowledge, this is the first time this effect has been isolated experimentally, as earlier measurements were either limited to small fluctuations around the stretched state 12 or suppressed the generation of excess friction through the attachment of large beads to the recoiling polymer end [13][14][15] . In the following, we elucidate the underlying physics of the enhanced friction and describe our dedicated set-up to measure it. We then present our measurement results and interpret them in the light of a systematic theory of nonequilibrium polymer dynamics 16 .
In semiflexible polymer dynamics, hydrodynamic friction arises both due to the polymer's transverse bending undulations and due to the contour's longitudinal motion with respect to the background solvent. For a weakly bending (stretched) polymer, longitudinal fluctuations are geometrically strongly suppressed relative to transverse fluctuations. The linear dynamics of transverse bending modes therefore not only dominates moderate transverse contour excursions, but also slow longitudinal fluctuations-for example, the dynamic longitudinal mean-square displacement of a monomer at late times t-N. But theory predicts that the short-time longitudinal dynamics, which becomes accessible by our dedicated experimental set-up, is strongly nonlinear and dominated by longitudinal drag. An example is provided by the initial stretching dynamics in response to a suddenly applied pulling force 10,[17][18][19] .
But such a fast stretching is limited by longitudinal drag for a relatively short initial phase of inhomogeneous tension propagation only. Sizable longitudinal motion also occurs during the reverse process of contraction, when the pulled-out wrinkles autonomously recover to their equilibrium level after the external tension is suddenly released-that is, during the initial stage of 'coiling up' 20 . This 'release' scenario, sketched in Fig. 1e, arguably g % 2pZL= logðL=dÞ þ g bead % 39 pN ms mm À 1 , tE1.7 ms. (d) Stretch relaxation (DNA-coated bead with one initially stretched polymer that is freely retracting): gE39 pN ms mm À 1 , tE5 ms (determined via f trap (t) ¼ f trap (0)/e). (e) The recoiling of a polymer from a stretched initial state is entirely driven by entropic forces or 'thermal noise' (red arrows). The additional contour length required to create the wiggles in the contour has to be pulled in from the polymer ends. The concomitant solvent flow (blue) exerts counteractive longitudinal drag forces that limit the maximum retraction speed and ultimately lead to the excess friction seen in scenario (d).
allows for the best experimental control over the comparatively scarcely investigated longitudinal motion limited by longitudinal drag, as it gives rise to an extended phase of quasistatic homogeneous tension relaxation limited by longitudinal drag 16,21 . A first glimpse of this type of motion-a power-law growth of the retraction or 'length deficit' with t 1/3 -has already previously been caught 22 . But with our set-up, which enables us to directly monitor the decay of the backbone tension, we can for the first time inspect the underlying physical mechanism. As we demonstrate next, the recoiling, although it is solely driven by entropy, viz., by the thermal forces resulting from the random bombardement of the polymer backbone with solvent molecules, involves such high local retraction velocities as to create the massive excess friction alluded to above (Fig. 1d).

Results
Measurements. Our dedicated set-up to reversibly and noninvasively stretch and release a strand of l-phage DNA is sketched in Fig. 2. A comprehensive description of the experimental procedures can be found in the Methods. Briefly, a calibrated optical trap embedded within a microfluidic cell serves to hold or relocate a single DNA-coated colloidal bead 23 . One of the grafted DNA molecules on the bead is pulled straight by applying an electrophoretic force with a nearby nanocapillary. After the electric field has abruptly been switched off at time t ¼ 0, a highspeed video camera tracks the bead displacement in the trap with submillisecond time resolution 24 . From the bead position, we infer the force f trap (t) exerted by the trap onto the bead. Up to the relatively small bead friction (that is fully included in our theory), f trap (t) is the value f P (L, t) of the backbone tension profile f P (s, t) at the grafted end s ¼ L. Typical time traces of the force f trap (t) exerted by the trap onto the bead are depicted in Fig. 3.
Theory. Our systematic theory is in good quantitative agreement with the data (Fig. 3, lines). Since the optically trapped bead moves by no more than E200 nm during the whole experiment, the bead-attached end can be thought fixed, thus closely approximating the ''release'' scenario described in refs. 16,25. Yet, taking also the slight bead motion into account, the theory allows us to reconstruct from f trap (t) the complete spatio-temporal evolution of the tension profile f P (s, t), including the tension f P (L, t) at the grafted polymer end, (Fig. 4) and the local retraction  Theory curves: f trap (t) including the dynamic backbone tension of the recoiling polymer and the bead drag (solid line), using a concentration-dependent persistence length of ' p ð20 mMÞ ¼ 62 nm, ' p ð50 mMÞ ¼ 58 nm, ' p ð100 mMÞ ¼ 55 nm, ' p ð500 mMÞ ¼ 44 nm, ' p ð750 mMÞ ¼ 41 nm (ref. 33), and a friction coefficient per length z k ¼ 0:65 mPas ; for comparison, the prediction for the hypothetical rigid-rod scenario from Fig. 1c (dotted) is also shown. At low salt concentrations (a) the surface charge of the capillaries has a significant influence on the ionic current 34 and the observed relaxation characteristics exhibit a larger variability. Alternative plots with theory curves for ' p ¼ 50 nm (independent of the salt concentration) are shown in Supplementary Fig. S1. A replication of the experiment using a different measurement protocol is documented in Supplementary Fig. S2. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2790 ARTICLE velocity along the polymer backbone (Fig. 5). In the taut initial state, the tension f P ðs; t o 0Þ f 0 P is constant throughout the polymer. Once the nanocapillary voltage has been switched off, the polymer end is force free, f P (0, t40) ¼ 0. After a fast tension propagating period that cannot fully be resolved with our set-up, the backbone tension f P (s, t) approaches a characteristic invariant spatial shape f Ã P ðsÞ, with slowly decaying amplitude (Fig. 4, see also the inset in Fig. 5 for a schematic illustration of the corresponding conformational dynamics). Notably, the force exerted by the polymer onto the trapped bead is predicted to be independent of the precise initial condition (and in particular of the precise value of the initial stretching force f 0 P , in good accord with our data in Fig. 3). According to Newton's third law the systematic variations in the backbone tension are balanced by the longitudinal viscous friction force acting along the polymer, from which the growth and decay of the retraction velocity along the polymer can be inferred 10,11,16,25 , as depicted in Fig. 5.

Discussion
The exact mechanism giving rise to the observed slow relaxation behaviour is intuitively best understood as a dynamical equivalent of the static worm-like chain force-extension relation. On a scaling level, we balance the effective friction force z k L@ t z (ref. 2) that retards the retraction of the end-to-end distance z of the molecule by the entropic driving force f P ¼ k B TL 2 =½4' p ðL À zÞ 2 that follows from the well-known Marko-Siggia expression for an almost straight conformation. Time integration then yields the aforementioned t 1/3 -growth of the deficit L À z and the asymptotic power-law decay f P ðtÞ$ðz 2 k L 4 k B T=' p Þ 1=3 t À 2=3 of the tension 25,26 . By allowing both the tension and the contraction to vary along the backbone, the argument can be made rigorous to yield the correct partial differential equation governing the evolution of f P (s, t) (refs 21,25). Further, including the optical trap and the bead friction, we obtain a slightly more complicated set of equations that can be solved numerically. To ease data analysis in future applications, we have extracted an approximate semiempirical formula from this first-principles theory (the relevant equations of motion and the definitions of w and a are given in the Methods section), This formula was validated against the exact numerical solutions which it matches closely over a wide range of polymer lengths L, bead sizes g bead , trap stiffnesses k trap , persistence lengths ' p and initial values of the tension f 0 P . It interpolates between the purely exponential retraction obtained for negligible excess friction (as, for example, in the limit of a very short/stiff polymer) and the algebraic retraction f P (t)Bt À 2/3 that would result for a rigidly fixed polymer end 25 (corresponding to the limit of a very stiff trap). Thereby, it provides a precise quantification of the friction enhancement anticipated in Fig. 1 and a very convenient starting point for further quantitative applications of our dynamic force spectroscopy setup. Both dimensionless parameters w and a are unambiguously defined in terms of experimental conditions (cf. Methods), thus leaving no free parameter save for the effective friction coefficient z k . The friction coefficient per length z k can be estimated asymptotically z k % 2pZ= logðL=aÞ $ 0:65 mPas (for a stretched molecule of diameter a ¼ 2 nm (ref. 27)), and we have used this value for all of our theory curves. This renders the comparison between data and analytical theory in Fig. 3 parameter free.
In summary, combining experiment and theory, we have developed a quantitative dynamic force spectroscopy set-up. We demonstrated that the internal dynamics of a recoiling taut DNA molecule generates a large excess friction that drastically delays the relaxation of an attached tracer bead, as quantitatively predicted by a simple but precise analytical approximation to the systematic theory. The theory also enabled us to reconstruct the complete spatio-temporal evolution of both the backbone tension and the rebound velocity along the polymer, from records of the tracer relaxation. In the future, it might be interesting to use this  Fig. 3. The computed velocity profile quickly settles down to an almost linear profile after the propagation layer has swept through the polymer (at tE1 ms). Yet, substantial retraction velocities persist near the free end, and the resulting enhanced friction (Fig. 1c) considerably retards the decay of the backbone tension at the grafted end. Inset: qualitative sketch (not to scale) of the recoil process and the accompanying change in backbone tension (from top to bottom). quantitative set-up to reveal how the characteristic dynamics is modified by the presence of DNA-protein interactions, nicks or supercoils 28 .

Methods
Experimental. The microfluidic cell consists of polydimethylsiloxane (PDMS) and encompasses two 100 ml reservoirs connected by a nanocapillary, whose orifice size defines the active sensing volume for single-molecule studies. We use quartz glass capillaries (Hilgenberg, Germany) with an outer diameter of 0.5 mm and a wall thickness of 0.064 mm (20 mM) and 0.1 mm (50-750 mM), respectively. At the bottom the microfluidic cell is sealed with a glass cover slide of 100 mm (20 mM)/ 130 À 160 mm (50 À 750 mM) thickness. The electric potential is applied with the help of two Ag/AgCl electrodes, one of them residing within the nanocapillary, the other located in front of the capillary tip. We use a potassium chloride (KCl) solution at concentrations of 20, 50, 100, 500 and 750 mM in 10 mM Tris buffer at pH 8.
Our custom-built optical tweezers set-up is assembled on an optical table and uses a 5-W ytterbium fibre laser (YLM-5-LP, IPG Laser, Germany) at a wavelength of l ¼ 1,064 nm (ref 24). Whereas the optical trap itself is static, the microfluidic cell is mounted onto a xyz piezo nanopositioning system (P-517.3 and E-710.3, Physik Instrumente, Germany). With a range of 100 mm in xy-and 20 mm in z-direction it allows for nm-precise manoeuvring of entrapped beads in relation to the surrounding cell. Illumination of the region of interest inside the sample cell is either done using a standard white light source (DC-950 Fiber-Lite, Edmund Optics, USA), or an optical fibre (100 W mercury arc lamp, LOT-Oriel, UK and 600 mm multimode silica fibre, NA0.39, Thorlabs, UK). In combination with a high-speed CMOS camera (MC1362, Mikrotron, Germany) this modular approach allows for real-time tracking of optically trapped colloids at up to 10,000 fps (frames per second) with 2 nm spatial resolution 24 .
A Faraday cage encloses the sample cell. The externally applied electric potential is held constant by a commercial amplifier (Axopatch 200B, Axon Instruments, USA) in voltage-clamp mode. Its headstage is mounted inside the Faraday cage and connected to the Ag/AgCl electrodes of the microfluidic cell. Concurrent measurements of the ionic current through the nanocapillary 29 allow the simultaneous trapping of multiple DNA strands to be ruled out.
Our DNA specimens are extracted from bacteriophage lambda (l-DNA, New England Biolabs, United Kingdom). We attach biotinylated l-DNA to 2.1 mm streptavidin functionalized polystyrene colloids (Kisker, Germany) 30 . With a binding constant of K A ¼ 4 Â 10 À 14 mol the interaction between biotin and streptavidin is almost covalent and hence suitable for long-term experiments 31 .
Before each experiment we determine the nanocapillary tip diameter via its current-voltage characteristics 23 . Afterwards, l-DNA-coated colloids are flushed into the microfluidic cell and the optical trap is calibrated by analysing its power spectral density 24,32 . The proper grafting of l-DNA to our polystyrene colloids is verified by repeating the power spectrum calibration for a number of particles.
Tension dynamics. Apart from a short-lived initial regime (tBns) that we cannot measure here, the relaxation behaviour of a freely contracting semiflexible polymer is governed by a generalization of the Marko-Siggia force-extension relation 2 to space-and time-dependent backbone tension f P (s, t) Our experiment ARTICLE For a freely relaxing chain, the above equation has been derived and solved systematically in the literature 16,25 . Within this theoretical framework, our stretching apparatus constitutes an initial condition f P (s, t ¼ 0), where the force gradient q s f P (s, t ¼ 0) is linearly proportional to the electric field and thus to the resistivity determined by the nanocapillary cross section, @ s f P ðs; t ¼ 0Þ / EðsÞ / 1 r cap: ðsÞ 2 : The fairly complex distribution of forces acting within the nanocapillary provides a good experimental approximation to a point force applied to the polymer end, as details of the initially applied tension profile f 0 P ðsÞ quickly diffuse away during tension propagation, see Fig. 4. Although in principle equation 2 breaks down close to the force-free end, and hydrodynamic interactions with the pore entrance or hydrodynamic boundary effects might locally increase the longitudinal friction coefficient z k , these finer points turn out to be negligible ( Supplementary Notes 1 and 2, Supplementary Figs S3 and S4).
We now extend equation 2 to dynamic force spectroscopy assays by coupling one polymer end to a linear trap of stiffness k bead and drag coefficient g bead . Given a certain time-dependent bead velocity v bead (t) that must agree with the local polymer velocity @ s f P ðs ¼ L; tÞ=z k at the bead-laden end, the dynamic tension profile is then determined as follows, The bead velocity v bead is not an external parameter but depends on the polymeric force f P exerted on the bead. As bead and trap together constitute a linear overdamped subsystem with characteristic relaxation time k trap /g bead , we can infer the bead velocity from the time-dependent polymer backbone tension evaluated at its bead-attached end f P (L, t), This closes equation 4, which we solve self-consistently by iterating in v bead (t) (Supplementary Methods).
To obtain a uniformly valid practical approximation to the resulting force relaxation f P (t): ¼ f P (L, t), we first reexpress the above system of equations in dimensionless form by measuring f P in units of f 0 P , distance s in units of polymer length L and time t in units of The equations of motion then read denotes the ratio between 'static' polymer drag and bead drag and is a measure of trap stiffness. Although the above equations may be solved to high accuracy using very little numerical effort (see Supplementary Methods), one should not underestimate the advantages of having a ready-made analytical expression for practical experimental work at hand. For this reason, we have devised the semi-empirical formula equation 1 that closely matches the result of the numerical integration, even in the nonasymptotic regime of initial decay, characterized by f 0 P ! f P ðL; tÞ \0:1 f 0 P . We now sketch its derivation.
For k trap pw-N, the bead-attached polymer end would not move at all, thus rendering the polymer dynamically equivalent to one half of a freely retracting polymer of length 2L. It has been shown before 25 that the latter scenario gives rise to a self-similar tension profile decaying like t À 2/3 at large times; as we infer from our numerical data, a regularized variant f P (L, t)B(1 þ 9t 2 ) À 1/3 provides a reasonable nonasymptotic fit at the cost of a mismatching prefactor at t-N. The opposite limit w-0 can be realized by g bead -N or L-0. In both cases, the relaxation is completely dominated by bead friction, thus yielding an exponentially relaxing force f P ðL; tÞ $ expð À t=tÞ with characteristic relaxation time ðz k L þ g bead Þ=k trap which, in units of t 0 , readst ¼ ð1 þ aÞ=w. For general values of a and w, we interpolate between both extremes using the superposition ansatz with a mixing parameter b, f P ðtÞ % b ð1 þ 9t 2 Þ 1=3 þ ð1 À bÞe À t=t : Next, we vary both w and a on a logarithmic scale, solving for f P numerically and fitting the above ansatz to the obtained solutions on the interval between f 0 P and f min % 0:1 f 0 P . To make sure we have covered all regions of interest, we verify that our data closely approaches the asymptotic solutions b(w-N) ¼ 1 (since w-N can be realized by taking k trap to infinity, leading back to the pure release scenario), b(a-0) ¼ 1 (directly follows from the dimensionless equations of motion) and b(w-0) ¼ 1/ (1 þ a). The latter asymptote corresponds to a virtually infinite persistence length ' p 4 4 L, such that the polymer retracts as a solid object of drag coefficient z k L and producing purely exponential relaxation on a timescalet as discussed above. In contrast, the timescale of internal contraction remains unchanged in units of t 0 such that tension propagation amounts to a sudden drop from the initially applied force f P ðt ¼ 0Þ=f 0 P ¼ 1 to some smaller force f P ð0 þ Þ=f 0 P ¼ b, which we identify with the force needed to drag along the polymer at an instantaneous speed _ xð0 þ Þ. As we have For large a, we do not have an analytic expression for finite w. However, as for any finite w, b must approach zero as a-N, we know that all nontrivial values of b are realized for very large values of w as a diverges. The boundary condition at the bead-attached end thus simplifies to @ s f P ð1; tÞ $ À a w _ f P ð1; tÞ:


extfieldsofstudy: Biology Medicine

text:
Structural Basis for Activity Regulation and Substrate Preference of Clostridial Collagenases G, H, and T*

Background: Bacterial collagenases degrade collagen substrates with high efficiency yet varying specificity. Results: The newly identified calcium site, aspartate switch, and conformational selectivity filter regulate substrate access to the active sites of these collagenases. Conclusion: The unanticipated dynamics of the substrate recognition sites plus zinc occupancy combine to tune the enzymatic activity. Significance: The crystal structures provide a rational framework to understand and optimize the isoform-dependent collagenase activities.

Clostridia employ collagenases for nutrition (e.g. saprophytic strains) and host colonization (1)(2)(3). They are large multidomain proteins of ϳ115 kDa belonging to the gluzincin superfamily of metalloproteases (4). They all share a common HEXXH zinc binding motif complemented by an additional glutamate located 28 -30 amino acids downstream, but the composition of their collagen recruitment domains varies. This can be exemplified by collagenase G (ColG) 4 from Clostridium histolyticum and ColA from Clostridium perfringens, both of which possess one polycystic kidney disease (PKD)-like domain and two collagen binding domains (CBDs), whereas ColT from Clostridium tetani lacks the former, and ColH from C. histolyticum possesses two PKD-like domains but only one CBD (3, 5, 6) ( Fig. 1). The recently determined crystal structure of ColG revealed a saddle-shaped architecture with a distinct segmentation of the N-terminal collagenase unit (Fig. 1). The collagenase unit is capable of degrading native collagen in the absence of the accessory domains within the C-terminally located collagen recruitment unit in vitro (7). It was further shown that the C-terminal saddle flap (Asp 398 -Asp 788 ) harboring the active site could cleave the peptidic collagenase substrate N- [3-(2-furylacryloyl)]-L-leucyl-glycyl-L-prolyl-L-alanine (FALGPA) but could not degrade collagen. Thus, the peptidases studied here have no collagenolytic activity. The domain nomenclature reflects the functional assignment ( Fig. 1).
Although vertebrate collagenases show a distinct substrate specificity and make only a single scission across all three collagen ␣-chains, bacterial collagenases hydrolyze native collagen at multiple sites and are capable of full degradation (3, 8 -9). Based on the ratio of the activities toward synthetic peptides and native collagen, collagenases from C. histolyticum were divided into two distinct classes (10 -12). Significantly, hydrolysis rates of ColG could be enhanced with substrates extending to the P4 residue, resulting in substrate cleavage rates by ColG comparable with those by ColH (12). The crystal structure analysis of ColG suggested that N-terminally extended substrates assist in their hydrolysis by employing additional interactions with the edge strand in ColG (7).
In addition to the active site zinc, clostridial collagenases were reported to require calcium for both their peptidolytic and collagenolytic activity (10,13). Atomic absorption spectroscopy indicated 6.8 and 5.1 Ca 2ϩ sites in ColG and ColH, respectively (10), but only the two calcium binding sites in the CBD were structurally elucidated (14). The lack of calcium binding in the ColG crystal structure can be explained by the presence of 0.1 M citrate in the crystallization buffer (7).
The interest in collagenases has been fostered by their multiple biotechnological and medical applications. For instance, the enzymes of C. histolyticum were recently approved as drugs to break down the tough cords in morbus Dupuytren (15)(16) and are widely used in human cell islet isolation (17,18) and wound debridement (19).
Here we present the crystal structures of the peptidase domains of ColH and ColT and the collagenase unit of ColG in the presence and absence of the peptidic inhibitor isoamylphosphonyl-Gly-Pro-Ala bound to the active site. The comparison of these homologues reveals differences in domain breathing motions and novel regulatory elements. Finally, we find that calcium and zinc are required for full peptidolytic activity, which can be explained by the spatial proximity of these metal binding sites in the peptidase domain.

EXPERIMENTAL PROCEDURES
Materials-All enzymes were purchased from Fermentas (St. Leon-Rot, Germany). Primers were obtained from Sigma-Aldrich; sequence analysis was performed at Eurofins MWG Operon (Ebersberg, Germany). All reagents were of the highest standard available from AppliChem (Darmstadt, Germany).
Cloning-Based on the crystal structure of the collagenase unit of ColG (Protein Data Bank entries 2Y3U, 2Y6I, and 2Y50) (7) and sequence alignments via ClustalW (20), we designed peptidase domain constructs of collagenase H from Clostridium histolyticum (Leu 331 -Gly 721 ; numbering according to Swiss-Prot entry Q46085) and collagenase T from C. tetani (Asp 340 -Lys 731 ; numbering according to Swiss-Prot entry Q899Y1). The peptidase domains were PCR-amplified from the respective full-length constructs (5) and cloned into a modified pET-15b vector, encoding for an N-terminal His 6 tag, fol-lowed by a tobacco etch virus protease recognition site (ENLYFQXGGT) and the enzyme-specific starting sequence.
Expression and Purification-The peptidase domain constructs of ColH and ColT were expressed in soluble form in Escherichia coli BL21(DE3) cells at 37°C in 2-liter baffled flasks in LB medium. The expression typically yielded 15 mg of pure protein from 600 ml of cell culture. All purification steps were carried out at 4°C. The three-step purification included immobilized metal affinity chromatography, tag removal, rechromatography, and size exclusion chromatography as a final polishing step, as described in more detail elsewhere (5,21). Protein expression and purification of the PKD-like domain FIGURE 1. Domain organization and quaternary architecture of mature clostridial collagenases. Clostridial collagenases are composed of two functional units, the collagenase and the collagen recruitment unit. The saddle-shaped collagenase unit is composed of an activator and a peptidase domain. The latter is further segmented into an upper (magenta) and lower half-domain (blue), flanked by a helper subdomain (cyan). The collagen recruitment unit consists of up to two PKD-like domains (yellow) and up to three CBDs (orange). The catalytic zinc ion and the catalytic residues are shown in a ball-and-stick representation (circled). In the figure, the peptidase domains of ColG, ColH, and ColT are superimposed. (Sub)domain boundaries of collagenase G, H, and T are given in tabular form (one-letter amino acid codes). For the CBD model, Protein Data Bank entry 2O8O was used.
constructs and of the collagenase unit were performed as described previously (5,7,22). Protein concentrations were assayed by detecting the absorbance at 280 nm. The molar extinction coefficients were calculated using the ProtParam tool available at ExPASy (23). The recombinant proteins migrated on a denaturing SDS-polyacrylamide gel with an apparent molecular mass of ϳ79, 45, and 9 kDa for the collagenase unit of ColG, the peptidase domains of ColH and ColT, and the PKD-like domain constructs, respectively, and were found to be at least 95% homogeneous.
Enzymatic Assay-Enzymatic assays with FALGPA as substrate were performed as described by van Wart and Steinbrink (13) and detailed in the manufacturer's protocol (AppliChem) with the following modifications: (i) to raise the buffer capacity, Tricine concentration and pH were increased to 250 mM and 8.2, respectively; (ii) calcium was omitted from the reaction buffer; and (iii) protein was used at final concentrations of 10 g/ml, respectively, in a 30-l reaction volume.
The individual FALGPA solutions were made from a 4.0 mM FALGPA stock solution prepared in reaction buffer, and the concentrations were verified in solution via UV absorbance at 305 nm (⑀ 305 ϭ 24.70 mM Ϫ1 cm Ϫ1 ). A 1.0 M solution of ZnCl 2 was prepared in double-distilled H 2 O, diluted 1:100 (10 mM stock) and 1:1000 (1.0 mM stock), whereby the latter was used for serial dilutions. A 5.0 M CaCl 2 and a 0.5 M EDTA (at pH 8.0) stock solution were prepared in double-distilled H 2 O and used for serial dilutions. All dilutions were done with reaction buffer. Protein samples used for enzymatic characterization were the same as for crystallization but at a lower protein concentration. Metal-depleted protein was prepared via incubation with 10 mM EDTA or 10 mM 1,10phenanthroline overnight at 4°C and rebuffered on a NAP-5 desalting column (GE Healthcare) pre-equilibrated with 250 mM Tricine, 400 mM NaCl, pH 8.2 (basic reaction buffer) before analysis. The protein stock solution of 0.1 mg/ml concentration was prepared in the reaction buffer (basic reaction buffer supplemented with the indicated CaCl 2 and ZnCl 2 concentrations) and incubated for 30 min at 4°C prior to the measurements. All measurements were carried out at 25°C, and the decrease in absorbance upon substrate cleavage was monitored at 345 nm in an Infinite M200 plate reader (Tecan).
All experiments were performed in triplicates and repeated at least twice. For K m and V max measurements, 14 different substrate concentrations were used ranging from 28 M to 3.5 mM. Initial reaction velocities were calculated using nonlinear regression (24). The data were fitted to the Michaelis-Menten equation by nonlinear regression analysis using GraphPad Prism version 5.00 software (GraphPad Software).
Crystallization, Data Collection, and Structure Determination-Crystallization conditions were screened by the sitting drop vapor diffusion method using various commercial screens at 20°C. Drops were prepared by mixing a 100-nl reservoir with a 200-nl protein solution containing, in the case of the peptidase domain constructs, 20 mg/ml protein in 10 mM Tris, pH 7.5, and 10 mM NaCl and, in case of the PKD-like domain constructs, 70 mg/ml protein in 10 mM Tris, pH 8.0, 25 mM NaCl, and 10 mM CaCl 2 . For pipetting, a Hydra II Plus One (Matrix Ltd.) liquid-handling system was used.
Crystals of the peptidase domain of ColH were obtained after 10 days in 20% PEG 3350 and 0.1 M sodium malonate, sodium formate, or sodium succinate, pH 7.0, and optimized by varying the initial crystallization conditions to 22.5% PEG 3350 and 0.15 M sodium formate at pH 7.25. Crystals of the ColT peptidase domain were obtained in 25% PEG 3350, 0.1 M HEPES, pH 7.5, and optimized to 25% PEG 3350, 0.1 M MES, pH 6.5, and 0.1 M NaCl. Crystals of the peptidase domains were harvested after cryoprotection by raising the PEG concentration to 30% in cryoloops, followed by flash cooling in a stream of nitrogen gas at 100 K.
Crystals were screened in house with respect to diffraction limit and transported to the beamlines ID14-4 and ID29 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) for high resolution data collection. The oscillation angle was set to 0.5 and 0.25°, and the exposure time was set to 1.0 and 0.125 s/frame, respectively. A total of 360 and 720 images were recorded and processed using iMosflm (25) and Scala (26) within the CCP4 suite (27). Data collection and processing statistics are summarized in Table 1. A polyalanine chain trace of the peptidase domain of ColG (Asp 398 -Gly 790 ; Protein Data Bank entry 2Y3U) was used for molecular replacement using the program Phaser (28). The model was iteratively improved by a combination of refinement using REFMAC5 (29) and manual building with COOT (30). The final stages of refinement were carried out by using bulk solvent correction, anisotropic scaling, and translation/libration/screw groups (31,32) to model large scale thermal motions. The progress of refinement was cross-validated throughout using R free with 5% of the unique reflections excluded from model refinement and map calculation. Refinement statistics are summarized in Table  1. Molecular figures were created with PyMOL (33). Contact areas were calculated by using the PISA server (34).
Protein Structure Quality-In order to understand the basis of the different enzymatic properties, we determined the crystal structures of the peptidase domains of ColH and ColT and the collagenase unit of ColG in the presence and absence of the peptidic inhibitor isoamylphosphonyl-GPA. The structures were determined in the resolution range from 1.7 to 2.2 Å with all residues being defined in electron density at excellent geometric and crystallographic parameters (Table 1). To verify the proposed calcium binding site in the PKD-like domain (22), the crystal structure of an N-terminally extended construct of the PKD-like domain of ColG (Asn 795 -Asn 880 ) was determined in the presence of 10 mM calcium at 0.99 Å resolution.
Relations of the Peptidase Domain within the Protein Structure Universe-A global structural comparison of the peptidase domain with deposited protein structures revealed that structurally related proteins all belonged to the MA-Clan; we found the highest agreement with members of the M1 (alanine aminopeptidase/leukotriene A4 hydrolase) and M4 (thermolysin) family (4); in absolute terms, however, the structural agreement was rather low, with 175-210 aligned residues and Z-scores of 6.8 -10.6, reflecting the low sequence identities of only 6 -14%. As a reference, the three collagenase homologues agreed to a Z-score of 50 or higher.
Importantly, we could identify structural homologues in the protein database only for the N-terminal (upper and lower) catalytic subdomain of the collagenase peptidase domain; by contrast, we could not detect a significant structural relation for the single C-terminal helper subdomain, suggesting a collagenase-specific role for the latter. These findings are in agreement with our previous structural analysis whereby the peptidase domain of ColG was segmented into a catalytic and a helper subdomain (7). The peptidase domains of ColH and ColT showed an analogous segmentation into a catalytic subdomain and a helper subdomain ( Figs. 1 and 2 (B and C)).
Partitioning of the Catalytic Subdomain-The catalytic subdomain can be divided into an upper N-terminal and a lower C-terminal half-domain, both of which embrace the active site cleft. As described analogously for ColG (7), inhibitor binding induced a 2-Å contraction of these segments in ColT (Fig. 2C). This conformational breathing emphasizes the hingelike active The helper subdomain is composed of a central ␤-sheet that is flanked by four ␣-helices (Fig. 2, B and C). It strengthens the relative arrangement of the catalytic segments within the peptidase domain. The stabilizing interactions of the helper subdomain with the upper segment and, to a lower extent, with the lower catalytic segment are reflected by their contact areas of 1060 and 418 Å 2 for ColG, 1046 and 347 Å 2 for ColH, and 955 and 503 Å 2 for ColT. The upper and lower segments form a 959-, 1115-, and 1359 Å 2 contact area, respectively. Consequently, the contact area between the upper and lower catalytic subdomains is larger by 42% in ColT than in ColG. Similarly, the lower catalytic subdomain has more than 20% stabilizing interactions with the helper domain, together suggesting a considerably more rigid quaternary arrangement within the peptidase in ColT as compared with ColG and also ColH. This difference might point toward different substrate specificities in these enzymes; alternatively, the PKD domain(s) present in ColG/H might compensate for the additional quaternary stabilization found in ColT.
The active site is positioned between the upper and lower catalytic segments. The characteristic HEXXH consensus sequence is located on the central helix; the third proteinaceous zinc ligand is a glutamate located 28 residues (ColG and ColH) or 30 residues (ColT) downstream on the neighboring gluzincin helix (Fig. 2, A and B, and supplemental Table 1). The spatial arrangement of the zinc ligands is identical in all three peptidases, notwithstanding the difference in sequential separation. The two zinc-coordinating histidines are stabilized by a conserved glutamine and glutamate positioned one helical turn downstream of the HEXXH motif and the gluzincin ligand, respectively (Fig. 4).
Three amino acids downstream of the third zinc ligand, an alanine side chain (ColG, Ala 558 ; ColH, Ala 490 ; ColT, Ala 502 ) serves as a hydrophobic basement ϳ4.4 Å underneath the catalytic zinc. Its position and strict conservation in clostridial collagenases suggest a pivotal role of the hydrophobic basement for the active site core and function, as described in great detail for metzincins (42). The S1Ј recognition site is formed by a conserved double glycine motif (Gly 493 -Gly 494 in ColG); its two amide nitrogens orient and arrest the P1Ј carbonyl oxygen in an oxyanion-type fashion, referred to as the secondary oxyanion pocket. Following the double Gly motif, Leu 495 -Glu 498 form the non-primed substrate recognition strand. This so-called edge strand participates in a mixed five-stranded ␤-sheet and is composed of mostly hydrophobic residues; Glu 498 anchors the edge strand by an ionic interaction with the calcium, as described below. The primed recognition site is delimited by a pronounced wall-like feature (Gln 511 -Phe 515 ) positioned on the loop preceding the central helix. The active site topology thus allows the P5-P3Ј substrate residues to bind in an extended conformation that is preferred for cleavage (43) (compare Fig. 3). In addition to these shared active site determinants, we identified several discriminating features around the active site of the three homologous peptidases ( Fig. 2A and supplemental  Fig. 1). The most striking difference within the uncomplexed structures was identified in the peptidase domain of ColH, where Asp 421 binds to the active site zinc (Fig. 4). Formally, this interaction mode is reminiscent of the aspartate switch in proastacin (44). This interaction was not observed in ColG and ColT, which have a serine at the position of the Asp 421 in ColH ( Fig. 2A). This ionic interaction completely rearranged the segment Asn 414 -Asn 424 but left the substrate recognition elements, such as the double-glycine motif (positions 425 and 426) and the edge strand (Met 427 -Glu 430 ), unaffected. Upon inhibitor binding, Asp 421 got displaced and adopted a conformation similar to that in ColG and ColT (supplemental Fig. 1).
Additionally, we found that the segment Lys 530 -Glu 536 in ColH folded into a prominent loop structure that selects access to the active site ( Fig. 3 and supplemental Fig. 1). The conformation of this selectivity loop is virtually identical in the complexed and uncomplexed ColH structure. Thereby, Tyr 531 and Phe 537 can interact with and stabilize other active site residues. This interaction results in a reaction tube-like compartmentalization of the active site. The combination of the aspartate switch and the selectivity loop renders the active site nearly completely inaccessible in the uncomplexed structure of ColH (Fig. 3). This selectivity loop was unique to ColH and may be related to the charged character of the corresponding segment in ColT and ColG, which possess one and three aspartate residues.
Peptidase Calcium Binding Site-Near the S5 site defined by the edge strand (Pro 499 ) and ϳ15 Å apart from the catalytic zinc, we could identify a structurally conserved calcium binding site (Fig. 4B). The calcium ion was octahedrally coordinated by two water molecules, three backbone oxygens (ColG, Ala 531 , Val 535 , and Gly 537 ), and a conserved glutamate side chain (ColG, Glu 498 ) (cf. Figs. 2A and 4). The latter partially coordinated the calcium in a bidentate manner, thus providing a seventh ligand to the calcium, albeit at a greater distance. Further details are provided in supplemental Table 2. In the ColG crystal structures, this site remained unloaded because of the presence of at least 125 mM citrate in the crystallization buffer. As evidenced by the comparison of Ca 2ϩ -unloaded (ColG) and -loaded (ColH/T) crystal structures, the liganding glutamate side chain undergoes a marked rearrangement upon Ca 2ϩ binding that allows Ca 2ϩ coordination. Due to its location  between the two catalytic helices, calcium binding braces the upper and lower half-domains of the peptidase and stabilizes thereby the zinc coordination.
Calcium Binding Site of the PKD-like Domain-Although the previously reported ColG crystals contained the PKD-like domain in addition to the collagenase unit, the PKD-like domain remained largely disordered (7). This disorder is presumably related to the presence of citrate in the crystallization buffer, which titrates away Ca 2ϩ ions. Because the crystal packing did not tolerate rebuffering to introduce calcium, we produced and crystallized an isolated PKD-like domain spanning residues Asn 795 -Asn 880 to investigate its calcium-binding properties. In this orthorhombic crystal form, we identified six proteinaceous ligands, namely Asn 795 , Lys 796 , Asp 825 , Asp 864 , and both of the carboxylate oxygens of Asp 823 , which together with a seventh water ligand coordinate the calcium ion in a pentagonal-bipyramidal geometry (Fig. 5). Detailed coordination geometries are summarized in supplemental Table 2. A previously described monoclinic crystal form of a PKD variant lacking four amino acids (Ile 799 -Asn 880 ) did not bind calcium (22). It is noteworthy that the overall structure and the behavior of the protein during expression, purification, and biophysical characterization remained identical; this indicated that calcium binding induces only local rearrangements at the N terminus of the PKD-like domain.
In addition, an N-terminally further extended PKD construct (Ile 792 -Asn 880 ) was crystallized in the same conditions as the Asn 795 -Asn 880 , but the additional residues (Ile 792 , Ser 793 , and Asn 794 ) could not be traced in the electron density, indicating their flexibility even in the presence of calcium (data not shown). Notably, in both PKD variants (Ile 792 -Asn 880 and Asn 795 -Asn 880 ), the calcium could not be extracted by incubating the respective crystals for up to 4 h with 100 mM EDTA.
Enzymatic Characterization-We selected the peptidase domain of ColT for enzymatic characterization in order to eliminate effects presumably caused by the aspartate switch (e.g. large variations of single measurements) in ColH, whereas ColG is the collagenase homologue with the lowest amidolytic activity (5). Even with ColT, we observed significant variations (cf. error bars in Fig. 6 and supplemental Fig. 2). After an extended series of kinetic experiments, we came to conclude that these variations reflect an important intrinsic property of this enzyme rather than an experimental error, as will be explained below.
The data showed that ColT could not be fully inactivated by metal-chelating reagents, such as EDTA or phenanthroline. Preincubation of ColT with phenanthroline irreversibly reduced the activity, albeit not completely (Fig. 6). Phenanthroline is thought to chelate and extract the catalytic zinc, which appears to be effective only in half of the proteases; once extracted, the active site cannot be reconstituted by adding metals (zinc, calcium, or a combination of both metals). Contrasting with the phenanthroline inhibition, EDTA reduced V max reversibly (Fig. 6). The effect is strongest when considering ColT, which was inhibited with EDTA in the reaction buffer, where V max was reduced to Ͻ25%. Importantly, each individual metal had only marginal effects, ϳ20% enhancement for Ca 2ϩ and ϳ0% enhancement for Zn 2ϩ (with errors overruling these effects). Only the simultaneous presence of both metals significantly increased V max by ϳ300% (Fig. 6).
These findings are puzzling at first sight but can be reconciled when assuming two different zinc-depleted peptidase conformations. The interplay of these conformational substates is described under "Discussion." It is noteworthy that the gel filtration-purified but otherwise untreated sample showed only 50% of the maximum velocity, indicating a partial loss of one or both metals upon size exclusion chromatography. Again, 10 mM Ca 2ϩ together with 10 M Zn 2ϩ could restore 100% activity, but neither of the metals alone had a significant effect.
Furthermore, metal extraction (via EDTA or phenanthroline) or addition had only small or insignificant effects on the K m (supplemental Fig. 2A). This suggests that metal extraction affects the active site geometry only locally, leaving major substrate recognition elements like the edge strand undisturbed (Fig. 4A and supplemental Fig. 1).

DISCUSSION
Domain Character of the Peptidase-The comparison of the isolated peptidase domains of ColH and ColT with the crystal structure of ColG containing the full collagenase unit plus the PKD-like domain reveals that the peptidase structure is unaffected by the contiguous domains (Fig. 1). This observation suggests that the peptidase is a true domain; this conclusion is corroborated by the facts that the peptidase (i) adopts a compact three-dimensional structure, (ii) is capable of independently folding, and (iii) can function as a peptidase independently of the rest of the protein.
Dual Function for the Helper Subdomain-The peptidase domain could be further segmented into the upper and lower half-domains, which adopt a thermolysin fold, and a C-terminal helper subdomain. The latter appears to stabilize the relative arrangement of the thermolysin-like half-domains and thus controls the breathing motion concomitant with substrate binding (Figs. 2C and 3 and supplemental Fig. 1). This interpretation is in line with the reported inactivity of a collagenase construct lacking the helper subdomain (5). The core of the helper subdomain resembles the structure of sheet B in the PKD-like domain (22). This structural relation suggests an additional function of the helper subdomain, whereby the helper subdomain acts as a platform to direct the three-dimensional arrangement of the downstream recruitment domains via a sheet extension mechanism. Such a mechanism was suggested to control the relative positioning within the collagen recruitment domains (7,22).
Structural Relation within ColG, -H, and -T Reflects Their Substrate Specificities and Preferences-Notably, the peptidase domain of ColG from C. histolyticum showed a higher structural similarity to ColT from C. tetani than to ColH from C. histolyticum. This structural relation is in particular evidenced by the extent of peptidase half-domain motions accompanying inhibitor binding and may, on a functional level, reflect the substrate specificities of the three homologous collagenases. The interaction areas between the inhibitor isoamylphosphonyl-GPA with the individual enzymes is largest in ColH (256 Å 2 ) Ͼ ColT (248 Å 2 ) Ͼ ColG (220 Å 2 ), as calculated using the PISA server (34). This relation explains the need of ColG for N-terminally extended peptides to obtain full peptidolytic activity (12).
Calcium and Zinc Are Both Required to Reconstitute the Activity of the Metal-depleted Peptidase-So far, only calcium binding sites within the CBD and the PKD-like domain have been identified (14,22). Although the calcium binding within the recruitment domains could explain their relevance for protein stability and interdomain arrangement, it could not account for its critical role in enzymatic activity, because the collagenase unit of ColG is capable of collagen degradation without its additional C-terminal domains (7). By contrast, the now identified calcium binding site within the peptidase domain is in 15-Å proximity to the active site, (Fig. 4A and supplemental Fig. 1). Upon calcium binding, the upper and lower segments of the peptidase domain are clamped together, stabilizing the conformation of the ligands around the catalytic zinc ion. Conversely, the catalytic zinc tethers the peptidase half-domains and thus preforms the calcium binding site within the peptidase domain. Within this structural framework it is understandable how zinc and calcium can cooperate in activity enhancement, as observed in Fig. 6. Nonetheless, some of the observed effects may appear counterintuitive at first sight, including the role of zinc; the addition of zinc with or without calcium could not restore the activity of phenanthrolinetreated ColT, suggesting that zinc could not be reconstituted. By contrast, zinc in addition to calcium is crucial to recover the activity of EDTA-treated ColT. These seemingly contradictory effects can be reconciled when proposing two distinct zincdepleted conformations, one being irreversibly collapsed (phenanthroline-treated) and the other one having zinc replaced by water with the zinc-chelating residues still in place, as observed in ColG (7). In the latter case, zinc was gently removed and replaced by a water molecule by citrate in the surrounding crystallization solution, with the geometry of the zinc-coordinating residues remaining in place. In this situation, the catalytic zinc can be reloaded, albeit only in the presence of calcium. By contrast, the phenanthroline-extracted zinc cannot be reconstituted. We propose that phenanthroline binds directly to the zinc in the active site. By doing so, it will disturb the zinc coordination sphere, which will help to extract the phenanthroline-bound zinc from the active site. Moreover, we propose that by the phenanthroline interaction, some of the zinc-coordinating residues will be irreversibly reoriented (e.g. the HEXXH motif, Glu 487 , or second layer residues Gln 462 and Glu 491 ; cf. Fig. 4).
The Aspartate Switch in ColH Stabilizes and Protects the Catalytic Zinc in the Absence of Substrates-Another surprising observation was that ColT is only partially inhibited when preincubated with an excess of phenanthroline or EDTA. Why would a complete inactivation require EDTA to be present in the reaction buffer? Again, we can only explain these phenomena when invoking different conformational substates. We could crystallize ColH in the absence of substrate analogues in a closed conformation, involving the aspartate switch Asp 421 (ColT; supplemental Fig. 1 and Fig. 4). Here, we found that Asp 421 coordinates the catalytic zinc in addition to the proteinaceous ligands (His, His, and Glu) present in all gluzincins, including ColG and ColT. Importantly, this closed conformation is loaded with both zinc and calcium but renders them inaccessible to the solvent. In particular, this conformation cannot be modified by phenanthroline, and we conclude that ϳ50% of the ColT population will adopt a closed conformation as crystallized and seen in ColH. It is further sensible to assume that the compressed conformation will also bind calcium more tightly, explaining why the same population should retain calcium even when occupied with EDTA.
The situation becomes more dynamic in the presence of substrates that will induce an open and active yet metal-vulnerable state. In this state, spontaneous metal bleeding will take place that is further driven by the presence of EDTA, explaining the steady activity reduction in the presence of EDTA during substrate turnover ( Fig. 6 and supplemental Fig. 2B). The overlay of different active site conformations with different metal contents can also explain the relatively large measurement variations.
Aspartate Switch in Related Zinc Proteases-The here described aspartate switch is reminiscent of a similar zinc coordination in proastacin (44). The comparison allows for some interesting conclusions as to the possible relevance of the aspartate switch. In particular, the comparison suggests an autoinhibitory mechanism that confers latency by blocking substrate access to the active site.
Although the formal parallelism with proastacin may support the autoinhibition hypothesis, the fact that ColH is a highly active peptidase supports the zinc stabilization hypothesis analogous to the tyrosine switch in active astacin (45). This notion is further supported by the contracted quaternary arrangement of the peptidase half-domains in ColH that is induced by binding of Asp 421 to the active site zinc (Fig. 1).
A further interesting analogy can be found when comparing open and closed states in ColH and ADAMTS4 (46). In ColH, both the active (open) and the closed states were crystallized in the presence of calcium, which is positioned at the nonprimed site. In ADAMTS4, the occupancy of the calcium near the active site correlates with the open conformation (i.e. calcium binding and the closed form appear incompatible in ADAMTS4) (46). This conformational reorganization is driven by the presence or absence of a substrate-like ligand binding to the active site, resembling the situation in ColH (and presumably also ColT, as suggested by enzymatic data), where the loop opening also appears to be induced by the substrate.
The active site of ColH remains rather restricted even in the inhibitor-complex structure with the Asp 421 displaced. This restriction mostly arises from a substrate selectivity loop that is characteristic of ColH and consistently explains the low collagenolytic activity, because triple helical substrates cannot easily access the restricted active site of ColH.
Preferences for Triple Helical Collagen Cleavage-On the one hand, the peptidase domains described here are insufficient to explain the collagenolytic properties of the full-length proteins, simply because they are unable to cleave collagen (7). On the other hand, we believe that the comparison of well characterized peptidolytic and collagenolytic activities of ColG and ColH point to structural features that are encoded already in the peptidase domain and discriminate their collagenolytic potency. Access to the active site is starkly restricted in ColH by a prominent "selectivity loop" as compared with ColG ( Fig. 3 and supplemental Fig. 1). This difference in accessibility correlates with the preference of single chain peptidic substrates over triple helical collagen in ColH versus ColG (10). However, an activator domain is required in addition to the peptidase domain for collagen cleavage. It is important to consider the extensive hydration shell around triple helical collagen, the structure of which will be stabilized by the water molecules. We further proposed that the activator and peptidase domains embrace the collagen substrate, thereby stripping many of the collagen-associated water molecules (7). This will lead to a destabilization of collagen and may in consequence lead to presenting vulnerable collagen sites to the peptidase active site.
Conclusions-The comparison of three clostridial collagenase structures allows us to rationalize distinct substrate specificities and to engineer desired peptidolytic activities. The disclosure of unexpected regulatory principles can be employed to tune the enzymatic activity for different applications. Among them, the most important adjustments can be gained by controlling the aspartate switch and the metal-loaded state. Further, the substrate selectivity filter should allow the modification of both peptidolytic and collagenolytic activities. Finally, the structures will also support the rational design of clostridial collagenase inhibitors.