extfieldsofstudy: Biology Medicine

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Genome-Wide Transcription Profiling of the Early Phase of Biofilm Formation by Candida albicans

ABSTRACT The ability to adhere to surfaces and develop as a multicellular community is an adaptation used by most microorganisms to survive in changing environments. Biofilm formation proceeds through distinct developmental phases and impacts not only medicine but also industry and evolution. In organisms such as the opportunistic pathogen Candida albicans, the ability to grow as biofilms is also an important mechanism for persistence, facilitating its growth on different tissues and a broad range of abiotic surfaces used in medical devices. The early stage of C. albicans biofilm is characterized by the adhesion of single cells to the substratum, followed by the formation of an intricate network of hyphae and the beginning of a dense structure. Changes in the transcriptome begin within 30 min of contact with the substrate and include expression of genes related to sulfur metabolism, in particular MET3, and the equivalent gene homologues of the Ribi regulon in Saccharomyces cerevisiae. Some of these changes are initiated early and maintained throughout the process; others are restricted to the earliest stages of biofilm formation. We identify here a potential alternative pathway for cysteine metabolism and the biofilm-associated expression of genes involved in glutathione production in C. albicans.

Medical advances associated with invasive procedures, aggressive immunosuppressive treatments for organ transplantation, and the widespread use of broad-spectrum antibiotics are the most important causes of nosocomial fungal infection. The opportunistic pathogen Candida albicans, the predominant species recovered from hospitalized patients (39), accounts for 10% of hospital-acquired infections; candidiasis is the fourth most common blood-borne infection with an attributable mortality of ca. 38% (14). In immunocompetent individuals, C. albicans occurs as a dimorphic commensal colonizer of mucosal membranes in the oral cavity, gastrointestinal tract, urogenital mucosa, and vagina; in immunocompromised patients, however, this organism can become pathogenic, resulting in proliferative growth on mucosal surfaces and systemically. One of the features that enhances C. albicans' pathogenicity and/or infectivity is its ability to grow tenaciously on the surface of medical devices and abiotic surfaces, as well as on mucosa. Mature biofilms are characterized by the production of a thick extracellular matrix and an altered resistance phenotype to common antifungal agents (11,12). The basis of the characteristic drug resistance of cells growing in biofilms is a source of much speculation, although recent studies suggest multiple factors involved in this process (5,53).
Biofilm formation proceeds through characteristic stages that define the process. It begins with the initial adherence of single cells to the substratum, followed by the formation of microcolonies which grow into a confluent but trabeculated monolayer. Once confluence is achieved, the network of yeast cells, pseudohyphae, and hyphae become encased within an extracellular matrix (50). The tenacity of biofilm adherence varies with cell type, growth conditions, and the properties of the abiotic surface. Mutants blocked in either hyphal or yeast morphology can form biofilms, but these differ in their adhesive properties from those produced by wild-type cells (6). The extracellular matrix secreted by cells growing as adherent biofilms differs from the material secreted by planktonically grown cells and from matrices associated with bacterial biofilms (5). Genome-wide transcription profiling has become an important and powerful tool in cell biology supporting the generation of testable hypotheses for novel processes not yet characterized at the molecular level. To characterize the initiating events in biofilm formation, we performed a detailed transcriptional analysis of the early stages of biofilm development in C. albicans using an Affymetrix oligonucleotide GeneChip representative of the entire genome of C. albicans (36,37). Contemporaneously, others were performing similar studies by using a PCR-based spotter array composed of 2,002 open reading frames (ORFs), chosen at random from the 6,419 ORFs encoding proteins of Ͼ100 amino acids in the C. albicans haploid ORF set (30). In that study (18), gene expression in mature, established biofilms (48 and 72 h) formed in yeast nitrogen base (YNB) under various conditions of flow, oxygenation, and glucose concentration were characterized; 317 ORFs were expressed independently of hyphal formation, whereas 86 were dependent on hyphal formation (18).
The analysis reported here begins with individual cell attachment and ends when the cells have attained confluence during the first 6 h of C. albicans biofilm formation, an interval in the process which differs from later mature stages (11). Our study also differs from that previously reported (18) with respect to conditions used for biofilm formation and the number of ORFs assayed, as well as in the methods of data analysis. We find striking differences in gene expression patterns between nonadherent and adherent cells within 30 min of surface contact; similar and unique patterns of gene expression are also evident in both biofilm and planktonic cells within these early stages of biofilm formation. Our data complement work on gene expression patterns in mature biofilms (18) and indicate that biofilm formation in C. albicans is accompanied by progressive changes in gene expression. Some of these changes are initiated early and maintained throughout the process; others are restricted to the earliest stages of biofilm formation.

MATERIALS AND METHODS
C. albicans strain and media. The genome sequencing strain SC5314 was used in these studies (20). Cells are routinely maintained at Ϫ80°C and plated on agar containing YPD (10 g of yeast extract, 20 g of Bacto peptone, 20 and g of dextrose/liter) before each experiment.
Establishment of biofilms. A single colony of C. albicans SC5314 was used to inoculate Ham's F-12 medium (Life Technologies, Gaithersburg, MD) and grown overnight at 30°C in a water shaker. Cells were centrifuged at 2,100 ϫ g for 10 min at room temperature and washed twice in fresh F-12 medium at 30°C. Cells at this point are designated as experimental condition time zero (T ϭ 0). For each time point, biofilms were formed in polystyrene petri dishes (100 mm by 15 mm [Fisher Scientific, Pittsburgh, PA]) using 10 7 cell/ml of inoculum in fresh F-12 medium (10-ml final volume per plate). Planktonic cultures were prepared by using the same inoculum size in 50 ml of F-12 medium in 250-ml polypropylene flasks, equalizing to the extent possible the surface/volume ratio in biofilm and planktonic cultures. Both cultures were incubated at 37°C in 5% CO 2 with rotation at ca. 50 rpm. After 30 min of incubation, medium from the biofilm plates was aspirated, pooled, and centrifuged, and the number of nonadherent cells was determined in a hemocytometer and used for RNA extraction; routinely, Ͼ80% of the cell inoculum was firmly attached to the abiotic surface within 30 min. Biofilm cultures were then thoroughly washed three times in prewarmed medium, and fresh F-12 medium added; plates were incubated for 6 h with constant rotation at 37°C and 5% CO 2 . A total of five time points were assayed for biofilms and equivalent planktonic cultures: T ϭ 30 min, T ϭ 90 min, T ϭ 150 min, T ϭ 270 min, and T ϭ 390 min. A set of biofilm plates corresponding to the five time points described above were stained with phloxine B dye (Sigma, St. Louis, MO), and propidium iodide and visualized by light microscopy.
AFM. Either cover glass or polystyrene disks (12 mm; Fisher) were submerged in 35-mm wells with 2.5 ml of F-12 medium. Biofilms were formed as described above. Each disk was washed twice with F-12 medium and dried at room temperature for 1 h before imaging. The disks were glued to a metal disk (12 mm) for magnetic attachment to the piezo-scanner of an atomic force microscope (Digital Instruments, St. Barbara, Calif.). The scan rates were ca. 1 Hz, with scan sizes between 15 and 60 m. All images were flattened manually in x and y directions by using the glass surface as the flattening reference (Fig. 1). Atomic force microscopy (AFM) facilitates highly accurate measurements of the topographic height (z axis of image), whereas the width of features is always subject to a tip-broadening artifact that derives from the shape of the AFM tip (26). We used height and width measurements of the long axis to more accurately describe the dimensional development of the blastoconidia during biofilm formation. The width measurements were corrected for the tip-broadening artifact by applying a tip angle of 35°for DNPS tips as described by Allen et al. (2).
RNA extraction. Before processing the complete set of samples, culture supernatants were collected for glucose determination (Glucose [HK] Assay Kit; Sigma, St. Louis, MO) and pH measurement. After each plate was washed twice with F-12 medium (37°C), cells were scraped from the plate and centrifuged at 2,100 ϫ g at room temperature, and RNA was immediately extracted. Planktonic cells were directly harvested by centrifugation as described above and processed concurrently. After collection by either scraping and/or centrifugation, cells were suspended in 3 ml of extraction buffer (0.1 M Tris-HCl [pH 7.5], 0.1 M LiCl, and 0.02 M dithiothreitol) and transferred to an ice-cold 50-ml tube containing 6 g of FIG. 1. AFM. All samples were imaged in the contact mode using a DNPS silicon nitride cantilever with a tip radius of ca. 10 nm (Nanosensors); the use of a porous membrane was not required (1). The scan rates were about 1 Hz with scan sizes between 15 and 60 m. acid-washed glass beads, 0.5 ml of 10% sodium dodecyl sulfate, and 5 ml of phenol-chloroform-isoamyl alcohol (25:24:1). The cells were disrupted by five cycles of vortexing (3 min each); between cycles the tubes were incubated alternatively for 3 min at 42°C and then at 0°C. The lysed cells were decanted and centrifuged at 10,000 ϫ g for 10 min at 4°C and subjected to two additional phenol-chloroform-isoamyl alcohol extractions before nucleic acid precipitation in 2.5 volumes of ethanol. Samples were stored at Ϫ20°C overnight, and RNA precipitates were collected at 10,000 ϫ g for 10 min. Total RNA was treated with RQ1 DNase (Promega, Madison, WI) for 1 h and cleaned by using an RNeasy Column (QIAGEN, Valencia, CA) prior to cDNA synthesis (Invitrogen, Carlsbad, CA); RNA samples were processed according to the manufacturer's instruction for Affymetrix GeneChips according to the protocol for 15 g of starting material. A total of twelve samples were obtained per experiment (one from time zero, five from biofilm, and five from planktonic and one from the nonadherent cells at 30 min). The experiment was performed in duplicate, and DNA microarray analysis was carried out from two independent sets of samples. DNA microarray design and analysis. A custom high-density oligonucleotide GeneChip manufactured by Affymetrix (Santa Clara, CA) was used; the characteristics of these GeneChips and experimental protocols with C. albicans samples are thoroughly discussed in Lan et al. (36,37). Gene annotations for the 7,116 open reading frames (ORFs) in the microarray and how they were assigned is described elsewhere (http://agabian.ucsf.edu). RNA labeling, target hybridization, washing, staining, and scanning were performed by using a GeneChip Hybridization Oven 640, a GeneChip Fluidics Station 400, and a GeneArray scanner (Affymetrix) as described previously (36). The technical reproducibility of the chips is very high due to their density, the random distribution of the probes within the matrix, and the synthesis of each probe directly on the substrate.
DNA microarray data analysis. GeneChips were scanned, and the resulting image files were used to calculate, normalize, and compare the hybridization intensity data by using the Microarray Suite 5.0 software (Affymetrix). The fluorescence signals of each array were normalized by global scaling with a target intensity of 500. The statistical algorithm within this software was used for the absolute analysis of each individual microarray. The single array analysis measures a relative level of expression of a transcript (signal) and determines the detection call indicating whether a transcript is detected (present [P]) or not detected (absent [A]). Absolute analysis of each microarray was followed by comparison analysis between two separate arrays; T ϭ 0 was selected as the baseline for comparison. This estimates the magnitude of fold change (signal log ratio) and the direction of the change (increase, decrease, and no change) in the expression of a transcript across the experimental time course for either biofilm or planktonic cultures. The data derived from each experiment was exported to Microsoft Excel, where the "signal log ratios" are converted to fold changes. For comparative purposes four variables were considered: (i) values with a no-change call, (ii) an absent signal with an increase-or decrease-change call, (iii) fold changes of Ͻ2, and (iv) intensity values of Ͻ100. If any of these criteria were positive in both planktonic and biofilm data sets, the probe set was excluded from analysis. The final results thus obtained consist of three different data sets: one set is described as the common gene set (CGS); genes in this set are upregulated or downregulated in both planktonic and biofilm cultures with respect to T ϭ 0. The second and third data sets contain the ORFs that are up-or downregulated differentially in either biofilm or planktonic cultures. The complete data set is available online (http://agabian.ucsf.edu).
Reverse transcriptase PCR (RT-PCR). Reactions were carried out by using SuperScript One Step RT-PCR with Platinum Taq (Invitrogen) and a PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA). A total of 1 ng of DNasetreated total RNA was used as a template in each reaction. Primers for each gene are included 5Ј-GAACACAAATGTCATGAAAC-3Ј and 5Ј-CAAGACTGAG GCACCAGC-3Ј for YWP1, 5Ј-GGTCACTTATTTACCTGATG and 5Ј-TGTG ATTCTACGTTCACCC-3Ј for MET3, and 5Ј-ACACCTGAGTTGACTCCA-3Ј and 5Ј-ACACCTGAGTTGACTCCA-3Ј for ORF19.6824. The reactions were performed with 1 cycle of 50°C for 30 min and 94°C for 2 min; 35 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min; and 1 cycle at 72°C for 10 min. PCR products were visualized in 1% agarose gels and in some instances qualitatively correlated with signals obtained from DNA microarray intensity data.

RESULTS AND DISCUSSION
In vitro biofilm model for C. albicans. The surfaces to which C. albicans adheres range from host mucosal membranes and skin to abiotic substrates used in medical devices. Coupled with C. albicans' ability to alter morphology and adapt to a range of environmental conditions, biofilm formation facilitates the transfer to and rapid proliferation of the fungus in the many anatomic sites that, in the immunocompromised host, support its growth. To approximate in vivo conditions, we used F-12 medium, 5% CO 2 , and 37°C; F-12 medium approximates human physiological conditions regarding fluid constituents, osmolarity, glucose concentration, and pH stability. Each of these factors, as well as the shearing force applied to the petri dish, critically influenced the rate of formation and the adhesive strength of these biofilms (15). These conditions are distinct from those used in a number of other reported biofilm studies (11,18). The kinetics of glucose consumption were essentially identical in both planktonic and biofilm cultures, with more than half of the glucose being consumed within 4 h (initial concentration of 10 mM) and entirely consumed after 6 h (data not shown). The pH of the medium was stable over the 6 h of the experiment, ranging between 7.6 and 7.8. Approximately 80% of the initial cell inoculum adhered to the petri dish within the 30-min attachment period.
AFM was used to visualize biofilm development. The AFM images shown in Fig. 1 were obtained from glass substrates but are representative of biofilm formation on polystyrene as well (data not shown). Image quality was enhanced when glass disks were used due to the reduced surface roughness of the glass versus polystyrene. AFM images of biofilms provided the opportunity to measure cellular parameters during the early phases of biofilm formation. Hyphal structure showed an approximately cylindrical shape, and the height measurement directly translates into a measurement of the diameter of the hyphae. In contrast, the blastoconidia is an ellipsoid structure with its long axis usually perpendicular to the hyphal axis. At T ϭ 30, C. albicans blastoconidia were on average 1.4 m high and 2.2 m wide, and germ tube development (length Ͻ 3 m) was evident. At the density of "seeding" of the biofilms used in these experiments, germ tubes attained lengths up to 10 m by T ϭ 90. At T ϭ 90, the beginnings of segmentation were evident; hyphae were generally straight and without evidence of branching or budding and the mother cells have increased to ca. 2.2 and 4.7 m in height and width, respectively. By T ϭ 150 a more intricate network was formed where most hyphae range in length from 20 to 30 m and form contacts either with neighboring hyphae or mother cells (57) and branching is initiated. The biofilm increased in density with time, and the AFM images at T ϭ 270 show a highly connected network of overlapping hyphae. At this time the pores or potential water channels in the network are reduced to Ͻ10 m in diameter. At T ϭ 390, the pore size is further reduced to an average of ϳ1 m; this pore size is maintained during continued biofilm development and retained in mature biofilms (data not shown).
After the initial attachment period at T ϭ 30 in biofilm development we observed what seemed to be a population of significantly larger and more elliptically shaped yeast form cells, reminiscent of the opaque phenotype of the WO-1 strain (56). Phloxine B, a stain that reflects alterations in cell membrane permeability, is used to differentiate white (W) and opaque (O) cells, as well as living and dead colonies in various yeasts (58). Staining of biofilms with Phloxine B produced a different pattern over the course of biofilm development: ca. 30% of the cells stained pink at T ϭ 30 and T ϭ 90 (Fig. 2), suggesting heterogeneity in the population of yeast cells attached to the substratum. However, there was no preferential staining of large versus small cells. At later times (T ϭ 270 and T ϭ 390), the proportion of stained cells decreased until only the tips of the hyphae took up stain (Fig. 2). Propidium iodide, which only stains cells with disrupted cell membranes, did not stain any of the cells throughout the experiment (data not shown), indicating that all cells within the biofilm remained viable for the 6 h of the study. Moreover, during the course of the experiment, biofilms grew as homogeneous hyphal cultures while the planktonic cells became progressively more hetero-geneous mixtures of hypha-pseudohypha-yeast forms (see below).
RT-PCR. Three differentially expressed ORFs, two biofilm (MET3, and ORF19.6824) and one planktonic (YWP1; cyclical expression pattern) were assayed by RT-PCR in addition to DNA microarray analysis. Comparisons of the RT-PCR products with DNA microarray signal values are shown in Fig. 3. MET3 transcripts are detected in both biofilm and planktonic cultures; however, at equal concentrations of total RNA and similar cycling programs, the amounts of product were substantially different in each case and consistent with the pattern obtained from DNA microarray analysis. YWP1 gene expression in planktonic cells is cyclical, both by DNA microarray analysis and RT-PCR, reflecting the initial transition from yeast to hyphal cells and the rapid reappearance of yeast forms in the culture. Conversely, YWP1 gene expression decreases in biofilm cultures and is essentially undetectable through T ϭ 390, both by RT-PCR and DNA microarray analysis, reflecting the hyphal nature of biofilms at this stage of development under our conditions. ORF19.6824, another gene upregulated in biofilm similarly confirms that the expression patterns are equivalent in both types of analysis.
Gene expression data. We assayed genome-wide changes in gene expression during the earliest stages of biofilm formation, beginning with the attachment of individual cells to the substratum through the formation of a contiguous cellular layer on the abiotic surface. The transcription data derived from these experiments was compared in several ways. "Vertical" comparisons were made during the time course (i.e., 1 h biofilm versus 1 h planktonic), similar to comparisons made in the study of gene expression in mature biofilms (18). We then went on and compared each vertical comparison longitudinally across the entire time course of the experiment. In these comparisons, genes that are equivalently induced as a function of time and/or morphogenesis in both biofilm and planktonic cultures are not scored since their values cancel each other; ORFs which display variable (i.e., cyclic) patterns of expression during the experimental time course were more difficult to compare across cultures, especially because the adherent early stage biofilms are essentially completely hyphal, whereas the planktonic cultures are heterogeneous with respect to morphologic types. In the "horizontal" comparison we analyzed changes in gene expression as a function of time within either biofilm or planktonic cultures, respectively, using the T ϭ 0 samples as a reference. These two data sets were then compared vertically with one another to identify genes that are regulated in both cultures and genes that are uniquely regulated in either. From these comparisons, a total of 2,492 ORFs whose expression varied during the course of the analysis were identified. Within this group, three different data subsets emerged: differentially upregulated genes in biofilm (41 ORFs; Table 1), differentially upregulated genes in planktonic cultures (25 ORFs; Table 2), and the remainder (2,426 ORFs) comprising a CGS (see Table S1 in the supplemental material) whose expression is similarly regulated in both types of cultures. The DNA microarray data set is derived from two independent experiments and showed a reproducibility of Ͼ95%. The genes expressed differentially in either biofilm or planktonic cultures were noted in both replicates. Only one representative set of data is presented here. Finally, we also com- pared the gene expression profiles of those cells that adhere to the substrate during the 30-min seeding period to the remaining 20% of the cells that did not adhere to the plates (nonadherent cells) (see Table S2 in the supplemental material).

Differential gene expression in biofilms.
A total of 41 ORFs were expressed more highly during the initial 6 h of biofilm development versus planktonic cultures; their relative levels of gene expression are found in Table 1. Of these 41 ORFs, nine (SUL1, MET10, HAL21, HAL22, MET14, MET15, MET3, ECM17, and ORF19.1159) encode proteins directly involved in sulfur metabolism. The changes in expression of this particular set of ORFs is striking and clearly indicate that not only particular ORFs but also the entire sulfur assimilation pathway is upregulated in early biofilm cultures. Moreover, the kinetics of this induction is also striking, beginning within 30 min of surface contact (Table 1 and Table S2 in the supplemental material) and increasing through the 6 h of the experiment. Unique to the present study and not detected previously (18) are C. albicans HAL21 and HAL22 ORFs, which are also highly upregulated in biofilm cells. HAL21 and HAL22 represent a gene duplication similar to that of HAL2/MET22 in Saccharomyces cerevisiae (45). C. albicans HAL21 and HAL22 encode a phosphatase that catalyzes the hydrolysis of either 3Ј-phosphoadenosine-5Ј-phosphate (PAP) and/or 3Ј-phosphoadenosine 5Јphosphosulfate (PAPS) to AMP and are thus likely to function in sulfur recycling. MET16, which encodes a 3Ј-phosphoadenylsulfate reductase that reduces 3Ј-phosphoadenylylsulfate to adenosine-3Ј,5Ј-bisphosphate and free sulfite, reported to be upregulated in mature (72 h) biofilms (18), is not upregulated during early biofilm formation. In the absence of concomitant upregulation of MET16, upregulation of HAL21/HAL22 suggests the activation of a futile sulfur cycle in biofilm cells. In plants, accumulation of toxic intermediates such as APS and PAPS is controlled by HAL2, which transfers the sulfur atom to an organic compound (47), typically glutathione (GSH) in most organisms (42,48). Functionally, GSH not only confers protection against reactive oxygen species but also serves as a storage and transport form of cysteine.
One of the most highly expressed genes in biofilm cells is MET3. The protein encoded by MET3 is the primary activator of sulfur assimilation in the cell. Studies in the fungal pathogen Cryptococcus neoformans indicate that MET3 also plays an important, but not understood role in its virulence and growth. MET3 expression levels in C. neoformans are not regulated by exogenous methionine, although they are in S. cerevisiae (64). In C. albicans, it is reported that MET3 transcription is fully repressed by 1 mM methionine or cysteine (10) and that repression of MET3 expression is detected at 0.1 mM cysteine or methionine. The F-12 media we use contain 0.2 and 0.03 mM cysteine and methionine, respectively, concentrations that are inhibitory for MET3 expression (10). As shown in Fig. 3, the levels of MET3 transcripts in planktonic cells are low, a finding consistent with repression of MET3 as described above. In contrast, even in the presence of what should be inhibitory concentrations of methionine and cysteine, MET3 is induced ϳ23-fold by T ϭ 30 in biofilm cells; thus, contact with the abiotic surface appears to override methionine/cysteine repression of MET3. The activation and maintenance of these pathways as a unique attribute in the transcriptome of cells growing as biofilms make these pathways an excellent drug target for the prevention of biofilm formation; one such drug already described elsewhere (3) might be azoxybacillin, a sulfite reductase inhibitor and natural antifungal agent produced by Bacillus cereus. The potential use of this drug will be the target of further investigation.
The identity of the ORFs induced in biofilm cells in the present study further suggests that sulfur assimilation in C. albicans differs substantively from that in S. cerevisiae and more   (46). Our assertion that this pathway is present in C. albicans is strengthened by the observation that, unlike S. cerevisiae, C. albicans met15-null mutants can grow on methionine-depleted media (61). We note that the C. albicans CysA and CysK homologues, ORF19.1159 and ORF19.7152, were not included/assayed in the previous study (18). Although the expression of both of these ORFs is upregulated only in biofilm cultures, there was no differential upregulation of MET6 (homocysteine methyltransferase) and SAM2 (S-adenosylmethionine synthase) suggesting again that the flow of sulfur under these conditions favors the synthesis of cysteine (Fig. 4).
Complementing the induction of sulfur assimilation path- ways is the upregulation of AGP3, an ORF that encodes a putative serine transporter protein and thus would provide a carbon acceptor for sulfur assimilation (7) through the OAS pathway. Similarly, OPT3, a probable oligopeptide transporter, based on its similarity to ISP4 of Arabidopsis, displays kinetics of expression similar to that of SUL1 during early biofilm formation ( Table 1), suggesting that OPT3 may play a role in the transport of sulfur-containing compounds (e.g., GSH or GSH-linked xenobiotics), as OPT1 does in S. cerevisiae (8). Also upregulated during early stages of biofilm development are NCE103, a carbonic anhydrase-like gene (22), and GCS1, a gene encoding ␥-glutamylcysteine synthase (4). Both of these enzymes are indirectly related to the sulfur pathway and oxidative metabolism. Mutant strains of NCE103 in S. cerevisiae are unable to grow under normal aerobic conditions, but complementation with the coding region of a carbonic anhydrase gene from Medicago sativa rescue this deficiency (13). In rats, carbonic anhydrase III also functions as a phosphatase upon glutathiolation of one of its cysteine residues (9). Gcs1, the rate-limiting enzyme in GSH biosynthesis, is associated with oxidative metabolism via the de novo production of GSH. In Schizosaccharomyces pombe, Gcs1 is under complex regula-tion by GSH feedback inhibition, the availability of L-cysteine, and the carbon source (32) and is associated with oxidative metabolism via the de novo production of GSH. Transcripts from both NCE103 and GCS1 are highly upregulated in biofilms; however, the kinetics of GCS1 induction differs from that of the sulfur assimilation pathways and NCE103. Although NCE103 is induced immediately upon attachment of cells to the abiotic surface, the signal values of the NCE103 transcript increase over time, and induction of GCS1 transcription is not observed until 4 h into biofilm growth. At this stage the cells have started to form a second layer and more intricate cell-cell interactions (Fig. 1); more than 50% of the available glucose has been consumed in both biofilm and planktonic cultures and the phloxine B staining pattern of the cells changes (Fig. 2). Taken together, these observations suggest that alterations in membrane structure and permeability and a requirement for GSH-related metabolic activities occur during the transitional period of the early to intermediate stage of biofilm formation. The potential role of GSH in the transient acquisition of drug resistance in biofilms merits some comment. In both mammalian tumor cells (51) and in the eukaryotic pathogen, Plasmodium falciparum (44), changes in GSH levels result in altered sensitivity of these cell types to a variety of drugs. Although this phenomenon remains to be explored in C. albicans, the genomic expression data support the hypothesis that, during biofilm development, alterations in GSH levels may play a role in the drug resistance phenotypes of cells growing as biofilms.
Of the remaining 28 ORFs upregulated in biofilm, 17 have homologues in S. cerevisiae, 5 of which are homologous to ORFs of unknown function; of these 17, 10 encode nuclear proteins (29). Not directly involved in sulfur assimilation but representing a significant second subset of genes expressed in biofilms are ORFs associated with phosphate metabolism: PHO89, PHO81, and ORF19.4424, a PHO2-like gene. In S. cerevisiae PHO89 encodes a membrane Pi transporter that mediates cation-coupled Pi transport acting in concert with a pH-dependent (alkaline) plasma membrane Na ϩ -ATPase (ENA1/Pmr2p) (17). A C. albicans homologue of ENA1, encoded by ORF19.5170, is preferentially upregulated in biofilm cells, along with the alkaline pH-regulated gene PHR1. Other homologues include ORFs associated with pseudohyphal growth (ORF19.5777); mitochondrial transport (YMC2); rRNA processing/Ribi regulon (KRR1, RRP8, and DIM1); tRNA modification (PUS7); an actin filament-binding protein (ABP140); an adenine deaminase (AAH1); and ORF19.7214 (similar to S. cerevisiae YBR056), a glucan 1,3-␤-glucosidase. Of the 28 ORFs differentially expressed in early biofilm formation (Table 1), 11 do not have a counterpart in S. cerevisiae; several of these, however, are similar to genes found in other organisms: ORF19.1797, which encodes a protein that contains a GutQ domain characteristic of many phosphosugar isomerases and phosphosugar-binding proteins required for bacterial lipopolysaccharide assembly (60); ORF19.4424, which encodes a protein with a domain similar to one found in the SurE survival protein of bacteria and is most closely related to an acid phosphatase of Yarrowia lipolytica; AMO2, which encodes a protein with similarity to a putative peroxisomal copper amino oxidase from A. niger (16); and SSP96, a protein containing a flavin-binding monooxygenase-like domain. ORF19.6675 displays a low degree of similarity to lipoic acid synthase, and ORF19.6824, a 467-amino-acid protein with a potential helix-loop-helix DNA-binding domain, is upregulated only in biofilm cells. ORF19.6824 appears to be specific for C. albicans, since Southern blot analyses with a collection of DNA from 11 different Candida species and S. cerevisiae failed to show positive hybridization, even at low stringency (data not shown). LDG3 encodes a leucine-aspartic acid-glycine rich protein of unknown function, which belongs to one of the largest gene families in C. albicans (A. Kuo, unpublished data); LGD3 is the only member of this gene family upregulated in biofilms. POL93, which is very similar to TCA8, encodes an RT that is upregulated nearly 100-fold at T ϭ 270 and is classified as a gypsy-like element with an internal primer site similar to a region of the Candida tRNA i Met (21). Differential gene expression in planktonic cultures. Although the cells growing as biofilms under the condition used are almost uniformly hyphal, as also observed in biofilm derived from RPMI1640 medium (10), equivalent planktonic cultures consist of a mixed population of hyphal, pseudohyphal, and yeast forms in F-12 media. This qualitative observation was quantitated by comparing the relative gene expression levels of HWP1 and YWP1, hyphal-and yeast-form specific genes, respectively (55). This dichotomy in population structure confounds interpretation of elevated levels of gene ex-  Table S1 in the supplemental material), suggesting distinct roles for these two almost identical (73% identity at the amino acid level) putative transcription factors. ORF19.5975 is a potential zinc finger protein and is similar to S. cerevisiae Adr1, a putative transcription factor, with respect to the position of zinc ligand and conserved DNA contact residues (52). The remaining nine ORFs displaying increased mRNA levels during the first 6 h of planktonic growth and which have no homologues in S. cerevisiae include: FMO2, a putative bacterial FAD-containing monooxygenase; CRW3, a potential cell surface antigen with a CFEM (for common in several fungal extracellular membrane proteins) domain present in proteins involved in fungal pathogenesis (35); SOD33, a protein likely similar to a secreted Cu/Zn superoxide dismutase from N. crassa; and six additional ORFs with unknown function ( Table  2). It is noteworthy that the expression of five ORFs in the planktonic category cluster with the yeast-form specific gene YWP1: ORF19.1482, ORF19.5975, CRW3, SOD33, and ORF19.633. Of the remaining five planktonic ORFs (of the twenty-five), four show constant fold changes through the entire 6 h, and one (INO1 inositol-1-phosphate synthase) is downregulated ( Table 2).

CGS.
The major change in both planktonic and biofilm cultures upon initiation of this protocol is the induction of hyphal morphogenesis. During the 6-h growth period of this experiment, a total of 2,426 ORFs were differentially regulated in both planktonic and biofilm cultures: 1,660 ORFs were upregulated, and 766 were downregulated. A total of 13% of the CGS ORF set (2,426) are ORFs of unclassified function, and almost 20% of the total CGS ORFs have no significant homology to any S. cerevisiae genome sequences (Table 3). Three different patterns of expression in the CGS were noted: group A, ORFs with initial low fold change values that increased over time (T ϭ 390/T ϭ 30, x Ն 2) (n ϭ 568); group B, ORFs with constant fold change values during the entire 6 h of the experiment (T ϭ 390/T ϭ 30, x ϭ 1) (n ϭ 1,120); group C, ORFs with initial increased fold change values that later decreased (T ϭ 390/T ϭ 30, x Յ 0.5) (n ϭ 738) (Table S1 in the supplemental material). Functional assignments of ORFs within the three groups were made using MIPS categories based on homology with S. cerevisiae genes (http://mips.gsf.de/proj/yeast/catalogues /funcat/index.html) (Table 3). Overall, the largest proportion of the CGS belongs to group B. Within the classes of gene expression profiles, for groups B and C the largest functional category is that of metabolism followed by protein fate, cellular transport, cell cycle, and DNA processing. Group C ORFs in the categories of cell cycle, DNA processing, and protein fate decreased at later times, as might be expected as the overall cell density increased in the cultures. There is a relative increase in the number of ORFs in group A in the metabolism category due to the expression of ORFs related to amino acid, nitrogen, and sulfur metabolism; this group also includes genes involved in cell rescue, defense, and virulence, including SAP5, SAP6, RBT5, ECM33, ACE2, ALS3, HWP1, TPK2; PLB3, PLB4, PLB5, and ORF19.1586, similar to a bacterial phosphatidylinositol-specific phospholipase C. A more detailed com- parison between groups C and A reveals that ORFs related to glycolysis and gluconeogenesis predominate in group C, whereas those associated with the oxidation of fatty acids and ionic homeostasis are upregulated in group A. ORFs with no homology to S. cerevisiae but which encode interesting amino acid motifs are also present in group A: for example, ORF19.5760 encodes a 392-amino-acid protein with a Gly-Ser domain at the carboxy terminal similar to that found in lustrin A, human and mouse cornified envelope loricrin, and the extracellular matrix protein keratin (54); ORF19.7104 encodes a mucin-like protein similar to microfilarial sheath protein Shp3 from the nematode Litomosoides (27).
Genes differentially expressed within 30 min of adherence. Adherence with a hard surface induces the expression of genes involved in the priming of plant fungal pathogens to signals that promote host invasion (33). Perhaps related to this phenomenon, C. albicans displays a thigmotropic response (63) hypothesized to assist the organism in foraging for suitable sources of nutrients (49) and, when pathogenic, in defining the route of tissue penetration (23). It has also been reported that when C. albicans encounters an abiotic surface the transcription of genes encoding efflux pumps MDR1 and CDR1 are rapidly induced, concomitant with the display of enhanced resistance to fluconazole (41). Given this context, we sought to determine whether contact sensing, a common feature in the biology of saprophytic fungi (49), results in a discrete change in gene expression in C. albicans. To answer this question, we analyzed alterations in the C. albicans transcriptome as a consequence of adherence to the abiotic surface by directly comparing gene expression values in T ϭ 30 adherent cells versus T ϭ 30 nonadherent cells (Table S2 in the supplemental material). Surprisingly, within the 30-min period allowed for attachment and adherence, the transcription of 554 ORFs, or nearly 9% of the genome, was altered by a factor of Ն2: 300 ORFs were upregulated in adherent cells. None of these contact-associated changes in gene expression correspond to those noted in the plant pathogen Colletotrichum gloeosporioises. Changes in the expression levels of the transporters CDR1, CDR2, and MDR1 were not observed; however, the experimental protocols used by Mateus et al. (41) and by us differ significantly. The most highly induced gene of known function in Candida biofilm cells is MET3 (ϳ23-fold at T ϭ 30). Roughly one-third of the 300 genes upregulated upon contact to the abiotic surface have no known function (see Table S2 in the supplemental material); of the remainder, ϳ57% are homologous to a subset of genes of the Ribi (for ribosome biogenesis) regulon of S. cerevisiae. This regulon consists of 236 genes primarily encoding proteins involved in ribosome biogenesis, subunits of RNA polymerases I and III, enzymes involved in ribonucleotide metabolism, tRNA synthases, translation factors, and proteins of unknown function predominantly localized in the nucleolus (31) and is coordinately and transiently downregulated in response to environmental or genetic perturbation (19,28,43). In C. albicans adhesion to polystyrene results in the upregulation at T ϭ 30 of a majority of the gene homologues in the S. cerevisiae regulon with the exception of genes encoding tRNA synthases and modifying enzymes, genes involved in nucleotide metabolism and translation initiation factors (see Table S2 in the supplemental material). Because the expression of members of this regulon is inherently unsta-ble (24), the levels of the mRNAs measured in our study may alternatively reflect a decrease in their rate of degradation. In general, the regulon is repressed by a wide variety of suboptimal growth conditions, by environmental factors that threaten cell viability, and by defects in the secretory pathway (19,38,43). Conversely, transcription of the regulon increases when starved yeast cells are provided with glucose (62) and is generally regulated by pathways such as those which coordinate cell size, progression through the cell cycle, and membrane integrity, with nutrient availability (31,38). Overall, expression of this regulon in S. cerevisiae seems to be associated with favorable growth conditions; its immediate induction in C. albicans upon adherence to the polystyrene surface may either provide C. albicans with the ability to rapidly respond to a change in environment and/or indicate that growth in the form of a biofilm is preferred.
Final remarks. Although the induction of sulfur assimilation pathways and the homologues of the Ribi regulon represent the most dramatic differences between planktonic and biofilm cells, involving the coordinate upregulation of functionally related subsets of ORFs, there are additional changes that are also likely to be important in understanding the process of biofilm development. Activation of enzymes involved in oxidative mechanisms such as NCE1O3 and GCS1, together with the expression of newly identified ORFs with no homology in the S. cerevisiae genome, creates new avenues to the search of new pathways during biofilm formation. Surprisingly, none of the genes implicated in azole resistance are upregulated during the first 6 h, but a potential role for GSH in drug resistance is a consideration. The challenge now lies in understanding the functional relevance of these major shifts in the readout of the genomic repertoire when cells grow as communities.


text:
Elevation of RNase R in response to multiple stress conditions.

Cells respond to adverse environmental conditions by synthesizing new proteins or elevating the levels of pre-existing ones that are needed to cope with the particular stress situation. We show here that Escherichia coli RNase R, a processive 3'-to5'-exoribonuclease, is dramatically increased in response to a variety of different stress conditions. Elevation of RNase R activity by as much as 10-fold was observed in response to entry into stationary phase, starvation, and cold shock, and a approximately 3-fold increase was seen during growth in minimal medium compared with rich medium. The elevation in RNase R activity was associated primarily with an increase in RNase R protein. RNase R was previously implicated in quality control of rRNA and tRNA and in the decay of mRNAs with extensive secondary structure. Its dramatic increase under multiple stress conditions suggests extensive remodeling of structured RNA in response to the altered environment.

The ability to sense and adapt to environmental change is critical to the survival of bacteria. Thus, upon encountering stress conditions, cells must rapidly alter their gene expression and remodel their RNA complement to deal with the changing environment. As a consequence, both new RNA transcription as well as RNA degradation must take place. However, while considerable information is available regarding changes in transcription in response to stress (reviewed in Ref. 1), very little is known about the RNA degradative machinery that comes into play under these conditions.
The types of stress with which cells must contend include deprivation of nutrients, changes in temperature, and exposure to noxious agents. For each of these conditions, rapid changes in mRNA content, and hence of mRNA stability, might be expected (1). In addition, under certain circumstances, such as starvation, degradation of stable RNA, i.e. rRNA and tRNA, also occurs (reviewed in Ref. 2). Stable RNAs, particularly rRNAs, account for the bulk of cellular RNA (3) and represent a potential storehouse of nutrients that could be tapped to help the cell survive a starvation condition. To do this, a change in ribosome structure and/or in RNase activity must occur that would render the usually stable ribosomes sensitive to degradation. The change in RNase activity could come about either by elevation of a pre-existing RNase or by synthesis of an additional RNase in response to the stress condition.
As part of an ongoing investigation of the functional role of RNases in Escherichia coli, we are examining how these enzymes respond to the imposition of a variety of stress conditions. In this paper, we focus on the processive, 3Ј to 5Ј exoribonuclease, RNase R (4,5). Our attention was drawn to this RNase because of its ability to digest rRNA and other structured RNAs in vitro (5,6) and to degrade rRNA fragments and REP sequences in vivo (6,7). We show here that RNase R levels increase dramatically in response to a variety of altered environmental conditions. A similar response to one of these stresses, cold shock, was recently reported (8). These data indicate that RNase R is novel among E. coli RNases and suggest that its action on structured RNAs may play an important role in helping cells cope with a changing environment.

Materials-[ 3 H]Poly(A) and poly(A)
were obtained from Amersham Biosciences and Sigma, respectively. Antibody against RNase R was prepared from purified protein (5) by Sigma-Genosys. Anti-rabbit IgG horseradish peroxidase conjugate, used as secondary antibody, was from Promega Biotech. All other chemicals were reagent grade.
Bacterial Strains and Growth Conditions-The E. coli K12 strain CAN20 -12E (RNase I Ϫ II Ϫ D Ϫ BN Ϫ ) and its derivative CAN20 -12ER Ϫ (RNase I Ϫ II Ϫ D Ϫ BN Ϫ R Ϫ ) have been described (4). These served as the RNase R ϩ and R Ϫ cells used in this study.
Cells were grown in liquid culture in either YT (yeast extract/tryptone) or M9/glucose (0.2 or 0.4%) media. Antibiotics, when present, were at the following concentrations: kanamycin, 25 g/ml; tetracycline, 12.5 g/ml. Normally, cells were grown at 37°C and were monitored by absorbance measurements at 550 nm. For experiments under various stress conditions, cells were first grown in YT or M9/glucose medium at 37°C prior to shifting to the experimental condition. Details are presented in the legends.
Media for starvation were as follows: carbon starvation, M9 without glucose; nitrogen starvation, M9/glucose lacking NH 4 Cl replaced with additional NaCl; phosphorus starvation, M9/glucose lacking Na 2 HPO 4 and KH 2 PO 4 replaced with additional NaCl and KCl and with 100 mM Tris-Cl, pH 7.4.
Preparation of Cell Extracts-Four ml of cells at ϳ1 A 550 (or the equivalent amount at a different absorbance value) were centrifuged and resuspended in 0.4 ml of solution containing: 20 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol, 300 or 500 mM KCl, and 0.1 mM phenylmethylsulfonyl fluoride. Extracts were prepared by sonication on ice using two 20-s pulses or five 10-s pulses, followed by centrifugation to remove cell debris. Protein concentration of the extracts was determined by the method of Bradford (9) using reagent purchased from Bio-Rad and bovine serum albumin as the standard.
Assay of RNase R-Activity of RNase R was determined by the release of acid-soluble radioactivity from [ 3 H]poly(A) as described previously (4). Assays were carried out in 50-l reaction mixtures containing: 20 mM Tris-Cl, pH 8.0, 0.25 mM MgCl 2 , 180 mM or 300 mM KCl, 40 g [ 3 H]poly(A) (50 -100 cpm/ nmol), and ϳ15 g of the indicated cell extract. Reaction mixtures were incubated at 37°C for the indicated amount of time. After precipitation with trichloroacetic acid, acid-soluble radioactivity was determined by liquid scintillation counting. One unit of RNase R activity converts 1 nmol of poly(A) to acid-soluble form/min.
Western Immunoblotting of RNase R-To determine the amount of RNase R protein present in extracts, proteins were resolved by 8% SDS-PAGE and transferred to a PVDF membrane by electroblottting. After blocking with nonfat milk (5%) in phosphate-buffered saline, RNase R was detected by incubating overnight at 4°C with a 1:10,000 dilution of anti-RNase R serum in phosphatebuffered saline containing 5% milk, 0.5% Triton X-100. Anti-rabbit IgG horseradish peroxidase conjugate was used as the secondary reagent and detection was with p-coumaric acid and Luminol. Underexposed films were used for quantitation. Titration with varying amounts of purified RNase R indicated that this procedure gave a linear response up to at least 150 ng of protein.

RESULTS
RNase R Levels during Growth-As an initial assessment of whether RNase R might be subject to regulation, we determined its activity as cells progressed from exponential phase through late stationary phase. The data presented in Fig. 1 show, in fact, that RNase R specific activity in extracts changes dramatically during cell growth. Thus, in the representative experiment shown, RNase R activity (filled triangles), which remains relatively constant during exponential phase, increases ϳ6-fold as the culture approaches stationary phase and increases an additional 2-fold during an extended stationary phase. In multiple experiments, the initial increase in activity varied from 4 -6-fold, whereas the increase in the second phase reproducibly was ϳ2-fold. That the increase in activity is actually all due to RNase R was confirmed by the absence of any activity change in extracts prepared from an RNase R Ϫ strain ( Fig. 1, empty triangles).
To determine whether the increase in RNase R activity was accompanied by an elevation of RNase R protein, Western immunoblotting with RNase R antibody was carried out on extracts derived from cells at the different growth phases (Fig. 1, filled circles). Quantitation revealed a ϳ3.5-fold increase (3-4-* This work was supported by Grant GM16317 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  fold in multiple experiments) in the amount of RNase R protein as the culture entered stationary phase concomitant with the major increase in RNase R specific activity. In contrast, no increase in RNase R protein was seen in the RNase R Ϫ mutant strain (Fig. 1, empty circles). Interestingly, in multiple experiments, no additional increase in RNase R protein was found as the culture progressed through stationary phase, a period in which RNase R specific activity doubles (Fig. 1). These data suggest that the second phase of increase in RNase R activity may occur by a different mechanism, perhaps protein modification. Further work with RNase R purified from stationary phase cells will be necessary to clarify this point.
It should be noted that in all experiments the initial increase in RNase R activity slightly exceeded the increase in RNase R protein. This was due to the fact that in extracts from all phases of growth RNase R activity is somewhat inhibited because of the presence of nucleic acids that compete with the [ 3 H]poly(A) substrate during the assay. The inhibition in extracts from early exponential phase (ϳ50%) was greater than that in extracts from early stationary phase (ϳ25%) resulting in an apparently greater increase in RNase R activity than actually occurred. Upon correcting for this effect, the increase in RNase R protein and increase in specific activity became essentially identical. Thus, these data show that RNase R levels are strongly influenced by growth phase.
Effect of Growth Rate on RNase R-Since a major reduction in growth rate accompanies the transition from exponential to stationary phase, it is possible that this slowdown in growth might be related to the observed increase in RNase R. To test this idea, cells growing rapidly in rich (YT) medium were compared with those growing more slowly in minimal (M9/glucose) medium with regard to RNase R specific activity and to levels of RNase R protein. The comparison was carried out with cells early in exponential growth to eliminate any effects due to stationary phase. As can be seen from TABLE ONE, cells growing at a slower rate in minimal medium were found to have ϳ3-fold higher RNase R activity and RNase R protein compared with those growing in rich medium. Based on these data, it is clear that growth rate does affect cellular levels of RNase R and, therefore, that the slowdown in growth could be related to the initial increase in RNase R seen during the entry into stationary phase.
Elevation of RNase R in Response to Starvation-A more extreme stress occurs under conditions of starvation during which cells cease growth. To determine whether starvation might affect the level of RNase R over and above that already seen during growth in minimal medium, cells were initially grown in M9/glucose medium until mid-exponential phase and then transferred after washing into the same medium devoid of either carbon, nitrogen, or phosphorus sources. After incubation at 37°C for 24 h under these conditions, cells were harvested and extracts assayed for RNase R activity (TABLE TWO). During the starvation period, the A 550 in cultures lacking carbon or nitrogen decreased somewhat, whereas in cultures lacking phosphorus, the A 550 increased slightly (see details in the legend to TABLE TWO). However, in multiple experiments, RNase R specific activity increased under each of the starvation conditions, amounting to ϳ1.4-fold for carbon starvation, ϳ2-fold for nitrogen starvation, and ϳ4-fold in the absence of phosphorus (TABLE  TWO) relative to the activity at the initiation of starvation. In RNase R Ϫ cells under these conditions, activity increased only slightly indicating that most of the increase observed in RNase R ϩ cells is, in fact, due to RNase R. It should be noted that nitrogen starvation of RNase R Ϫ cells led to a significant elevation of RNase activity, presumably due to an effect on another enzyme; however, that increase could not account for the much larger elevation in RNase R ϩ cells.
These data demonstrate that another stress condition, in this case, starvation, leads to an elevation of RNase R activity over that observed in minimal medium. Moreover, the fact that the level of increase in RNase R activity varies with the particular starvation condition (phosphorus starvation always was significantly higher) suggests that there may be a specific response to each type of starvation.
Elevation of RNase R in Response to Cold Shock-E. coli is known to respond to cold shock by increasing the synthesis of a variety of proteins that help it cope with this particular stress (10,11). In recent work (8), it was shown that RNase R protein was elevated ϳ7-fold following transfer of cultures from 37 to 10°C. To confirm and extend these observations to RNase R activity, cells growing at 37°C in rich (YT) medium during early exponential phase were rapidly transferred to several lower temperatures. At various times after the temperature downshift, cells were harvested for measurement of RNase R activity and RNase R protein.
The data in Fig. 2 show that upon transfer of cells to 10°C, RNase R activity is elevated ϳ10 -12-fold. The increase is already apparent at 1 h and reaches a maximum at 3 h after the temperature shift. RNase R activity is maintained near the maximum level to at least 6 h. Cells cease growth under these conditions. No change in activity was observed in extracts from RNase R Ϫ cells (Fig.  2) confirming that the elevated activity is due to RNase R. Western analysis revealed that RNase R protein was elevated at 10°C as well, to a maximum of ϳ5-fold in multiple experiments (data not shown). It is not yet clear whether the ϳ2-fold difference between the increase in activity and the increase in protein is meaningful.
Significant, but smaller, increases in RNase R activity also were observed at 15°C and 25°C (data not shown). At 15°C, the increase still was considerable, ϳ8-fold; however, at 25°C, only ϳ2-fold elevation was obtained in multiple experiments during a 6 h time period. It should be noted that cells are able to grow at these latter two temperatures. These data clearly demonstrate that cold shock is an additional stress which leads to dramatic elevation of RNase R protein and activity and that the more severe the cold shock the more pronounced the effect on RNase R.
Effect of Heat Shock on RNase R-In contrast to the dramatic elevation of RNase R during cold shock, the effect of heat shock was much more modest. Shifting the growth temperature from 37 to 42°C led to little, if any, increase in RNase R specific activity. However, upon shifting to 45°C, a reproducible elevation of ϳ2-fold was observed in multiple experiments after 90 min at the elevated temperature (data not shown). No increase was observed in RNase R Ϫ cells. However, since cell growth is slowed somewhat at 45°C compared with 42°C, it is not yet clear whether the small elevation of RNase R at 45°C is a specific response to the heat shock or is due to the reduction in growth rate.

DISCUSSION
The data presented here demonstrate that RNase R activity in E. coli is elevated in response to a variety of altered environmental conditions. These include entry into stationary phase, minimal versus rich media, starvation for carbon, nitrogen, or phosphorus, cold shock, and perhaps, heat shock. It is not FIGURE 1. Response of RNase R to growth phase. Cells were grown in YT medium at 37°C to the indicated A 550 and samples withdrawn for RNase R assay and determination of RNase R protein as described under "Experimental Procedures." Filled symbols, R ϩ cells; empty symbols, R Ϫ cells; squares, A 550 ; triangles, relative RNase R specific activity; circles, RNase R protein. RNase R specific activity and protein of the RNase R ϩ cells in early exponential phase were set at 1 for ease of comparison. The actual specific activity at this point was 3.1 units/mg of protein. Influence of growth rate on RNase R Cells were grown to early exponential phase in either rich (YT) or minimal (M9/glucose, 0.2%) medium. Samples were taken for determination of RNase activity and RNase R protein as described under "Experimental Procedures."

YT M9/glucose
Doubling time (min) a 32 74 Relative RNase R specific activity a 1.0 2.5-3.0 Relative RNase R protein a 1.0 ϳ3 a RNase R specific activity and protein in YT medium were set at 1.0. RNase R specific activity in YT medium was 5.6 units/mg. The values shown are averages or the range of two to five experiments, depending on the measurement.
yet clear whether a common physiological signal underlies the response to all of these diverse environmental stresses or whether multiple signals might be involved. One piece of evidence suggesting the possibility of multiple signals is the finding that starvation, for either carbon, nitrogen, or phosphorus, elevates the level of RNase R severalfold over and above the increase already seen in minimal medium when compared with rich medium. For example, during phosphorus starvation, the overall increase in RNase R specific activity is close to 10-fold when compared with cells growing exponentially in rich media. Similar increases were observed during stationary phase and during cold shock. Thus, although further work will be necessary to unravel the details of the regulatory mechanisms that result in elevated RNase R, it is already clear that the magnitude of the increase in RNase R activity is quite dramatic and novel when compared with anything seen previously with respect to physiological regulation of RNases. In a recent study by Cairrao et al. (8), it was also found that RNase R protein (RNase R activity was not determined) is elevated 7-8-fold upon cold shock at 10°C. In that work, it was suggested that the elevation of RNase R is due to stabilization of the rnr message during cold acclimatization and that subsequently the message is regulated by polynucleotide phosphorylase (PNPase). 2 However, the mechanism resulting in the initial stabilization of rnr message and the signal inducing the stabilization upon cold shock were not identified. RNase R has also been found to be regulated ϳ3-fold during the cell cycle of Caulobacter crescentus (12). However, the mechanism of this regulation also is not understood. Nevertheless, based on these observations, and those reported here, it is clear that RNase R is subject to extensive physiological control of its expression and its activity. Thus, not only do RNases affect the maturation and stability of cellular RNAs through their catalytic properties, they themselves are regulated by environmental and cellular conditions. The large increase in RNase R specific activity in response to the various stress conditions raises the question of what functional role elevated RNase R might serve in these situations. RNase R is unique among E. coli exoribonucleases in its ability to digest through extensive secondary structure by itself (4,6). In this regard, it is complementary to PNPase, which also digests through secondary structure as part of the degradosome (e.g. ref. 13 and 14). In the absence of both of these exoribonucleases, E. coli cells are inviable (4, 7) implying that they are solely responsible for digesting structured RNAs. Interestingly, in a psychrotrophic bacterium, Pseudomonos syringae Lz4W, RNase R is part of the degradosome (15).
RNase R has been shown to play a major role in degradation of REP sequences in mRNA (6) and in quality control of rRNA (7) and of defective tRNA (16). 3 Each of these substrates contains extensive secondary structure. Thus, based on its in vitro catalytic properties (5) and its already known in vivo functions, it is most likely that elevated RNase R would be used to help degrade structured RNAs under stress conditions. It has been suggested that RNase R plays a role in maturation of tmRNA during cold shock (8). However, we consider this unlikely as the processive nature of RNase R makes it ill-suited to function as a maturation RNase. Moreover, in Caulobacter, its action on mature and precursor forms of tmRNA is degradative, and it is not used for maturation (12). In fact, RNase R has not yet been shown to participate in the maturation of any RNA molecule. Rather, all its known functions are in degrading RNAs (6,7,16).
Considering this information, and the fact that rRNA, the major cellular RNA, is known to be degraded during stationary phase and starvation (2), we speculate that elevated RNase R is needed for this process. Perhaps, during cold shock, a significant portion of newly made ribosomes are misassembled, and RNase R is required for their destruction as well, akin to its known role in rRNA quality control (7). Inasmuch as PNPase also is known to participate in rRNA quality control (7), it is interesting that this enzyme also is elevated during cold shock (17)(18)(19). Whether PNPase is similarly elevated in stationary phase and during starvation is to our knowledge not known. It would be of interest to examine this point.
Based on the information presented here, RNase R appears to be an important stress protein, and consequently, RNA degradation may be an important component of the stress response. Examination of the mechanism of its regulation during stress and of the consequences of its absence under stress conditions should greatly increase our understanding of the stress response and of the importance of RNA turnover for this process. Effect of cold shock on RNase R activity. Cells growing in YT medium at 37°C were rapidly transferred at early exponential phase to 10°C. At the indicated times, samples were withdrawn for measurement of RNase R activity and RNase R protein (data not shown) as described under "Experimental Procedures." RNase R specific activity is presented relative to the specific activity at the time of the temperature shift, which is set at 1. The actual specific activity at this point was 5.6 units/mg of protein. Depending on the time point, data are from one or are averages of up to three experiments.

Effect of starvation on RNase R activity
Cells were grown in M9/glucose(0.4%) medium. At an A 550 ϳ0.2, cells were harvested, washed two to three times with the appropriate starvation medium (see "Experimental Procedures"), and then suspended in the starvation medium at the original cell density. After 24 h at 37°C, cells were harvested for RNase R assays. The A 550 of the cultures decreased ϳ50% during carbon starvation, decreased ϳ20% during nitrogen starvation, and increased ϳ35% during phosphorus starvation.

Strain
RNase


text:
Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone

Contrary to the results of direct expression, various human proteins (ferritin light-chain, epithermal growth factor, interleukin-2, prepro-ghrelin, deletion mutants of glutamate decarboxylase and arginine deiminase, and mini-proinsulin) were all soluble in Escherichia coli cytoplasm when expressed with the N-terminus fusion of ferritin heavy-chain (FTN-H). Through systematic investigations, we have found that a specific peptide motif within FTN-H has a high affinity to HSP70 chaperone DnaK, and that the peptide motif was composed of a hydrophobic core of three residues (Ile, Phe and Leu) and two flanking regions enriched with polar residues (Gly, Gln and Arg). It was also observed that all the recombinant proteins expressed with the fusion of FTN-H formed spherical nanoparticles with diameters of 10–15 nm, as confirmed by the transmission electron microscopy image. The protein nanoparticles are non-covalently cross-linked supra-molecules formed by the self-assembly function of FTN-H. Upon the formation of the supra-molecule, its size is likely to be limited by the assembly properties of FTN-H, thereby keeping the self-assembled particles soluble. This study reports on the dual function of FTN-H for fusion expression and solubility enhancement of heterologous proteins: (i) high-affinity interaction with DnaK and (ii) formation of self-assembled supra-molecules with limited and constant sizes, thereby avoiding the undesirable formation of insoluble macro-aggregates of heterologous proteins.

INTRODUCTION
Ferritin plays a central role in iron storage and in the maintenance of intracellular iron balance for the growth and survival of most organisms (1)(2)(3). The functions of ferritin suggest that it may serve as a cytoprotective protein, minimizing oxygen-free radical formation by sequestering intracellular iron (4). Ferritin molecules isolated from vertebrates are composed of two types of subunits (heavy chain and light chain), whereas those from plants and bacteria contain only heavy chains. Mammalian heavy chain and light chain show $54% sequence identity. In mixed subunit 24mer (heteropolymers) with a spherical shell structure, heavy (H) and light (L) chain subunits have similar conformations of principally four a helix bundles (5)(6)(7)(8). Four years ago, the Korean Food and Drug Administration prohibited the commercial manufacture of animal ferritin (from equine spleen extracts), which had been used to treat human iron deficiency, because of its probable complication of virus infection. Thereafter, the production of recombinant human ferritin in Escherichia coli has been used as a possible substitute for equine ferritin extracts.
It was reported that contrary to ferritin L-chain (FTN-L), ferritin H-chain (FTN-H) and H-L hybrid (FTN-H fused at the N-terminus of FTN-L) are soluble in E.coli cytoplasm, probably due to their enhanced folding efficiency. The recombinant ferritin H-L hybrid as well as the H-chain showed the same iron storage activity as natural copolymers of human ferritin (9). Various human proteins [epidermal growth factor (EGF), interleukin-2 (IL-2), prepro-ghrelin (ppGRN), a deletion mutant of glutamate decarboxylase (GAD 512-585 ), a deletion mutant of arginine deiminase (ADI 132-410 ) and minipro-insulin (mp-INS)], forming inclusion bodies upon direct expression, were all soluble in E.coli cytoplasm when expressed with the N-terminus fusion of ferritin H-chain. Considering the structural diversity of the above human proteins *To whom correspondence should be addressed. Tel: +82 2 3290 3304; Fax: +82 2 926 6102; Email: leejw@korea.ac.kr The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org fused to ferritin H-chain, it seems reasonable to assume that the de novo folding of proteins with ferritin H-chain fusion is not determined by direct protein-protein interaction between FTN-H and the target protein, but instead, the assistance of a global folding enhancer, a molecular chaperone, is much more important. Misfolding and aggregation of proteins are major damaging consequences of stress situations, such as heat shock and pathophysiological states (10)(11)(12). The central cellular defense against such damage is a molecular chaperone, which prevents aggregation, assists in refolding and mediates the degradation of misfolded proteins (13,14). Chaperones can cooperate in vitro as part of a functional network, in which 'holder' chaperones prevent the aggregation of misfolded proteins, whereas 'folder' chaperones actively assist in refolding (15,16). The Hsp70 chaperone DnaK participates in the folding of a subset of newly synthesized proteins, prevents protein aggregation in a denatured condition and refolds misfolded proteins.
Recently, Vandenbroeck et al. (17) identified conserved DnaK-binding sites in the N-and C-terminal halves of helix B and C of diverse 4-helix-bundle proteins, respectively, that belong to the superfamily of interferon-g/interleukin-10-related cytokines. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces exhibiting the strongly hydrophobic character of the interior core of a single-chain folded domain. They proposed that the binding of DnaK may constitute the hallmark of a novel conserved regulatory mechanism, in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric 'folding' interface. Hidaka et al. (18) also found that the propeptide of guanylyl cyclase, which activates peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, has a dual function in the proper folding of a mature peptide. This means that the N-terminus propeptide of pro-GCAP-II is critical, not only for disulfide-coupled folding but also for the net stabilization (or dimerization) of pro-GCAP-II.
In this study, diverse recombinant proteins, commonly with FTN-H fusion, were observed to form stable homopolymeric supra-molecules without the formation of insoluble protein aggregates. Seemingly, the supra-molecule size is limited by assembly properties of FTN-H, thereby keeping the supra-molecules soluble. The target proteins localized inside the spherical supra-molecules might have been in a protective environment against undesirable protein aggregation. We also identified a specific DnaK-binding motif within FTN-H and demonstrated the efficacy of FTN-H as a potent solubility enhancer upon heterologous protein expression in E.coli.

PCR-based site-directed mutagenesis
Site-directed mutagenesis for preparing various mutants of FTN-H and light chain and glutathione S-transferase (GST) was performed according to the standard protocols for overlap PCR (19). PCRs were performed in a final volume of 20 ml with the following reagents: 20 mM Tris-HCl, pH 8.3, 100 pmol of each primer, 200 mM dNTPs, 1.5 mM magnesium chloride and 2.5 U Taq polymerase (BRL). PCR cycling was performed as follows: initial denaturation at 94 C for 120 s and then 20 cycles of 94 C (45 s), 60 C (60 s) and 72 C (120 s).
After the completion of 20 cycles, the reaction mixture was incubated for 5 min at 72 C. The products were gel purified after agarose gel electrophoresis.

Gene expression, purification of recombinant proteins and bioactivity assay
For the shake flask experiments (250 ml Erlenmeyer flasks, 37 C, 200 r.p.m. Shaking Incubator IB-3100, New Power Eng., Korea), LB media containing 100 mg kanamycin (or both ampicillin and kanamycin where applicable) per liter of culture was used. When the culture turbidity (OD 600 ) reached 0.5, the gene expression was induced with the addition of isopropyl-b-D-thiogalactopyranoside (IPTG) (0.5 mM). After further 3-4 h cultivation, the recombinant cells were harvested by centrifugation (6000 r.p.m. for 5 min) and the cell pellets were resuspended in 5 ml distilled water. Cell disruption was done by using Branson Sonifier (Branson Ultrasonics Corp., Danbury, CT). The cell-free supernatant and insoluble protein aggregates were separated at 13 000 r.p.m. for 10 min. The isolated inclusion bodies, if any, were then washed twice with 1% Triton X-100. The cell-free supernatant and the washed inclusion bodies were subject to PAGE analysis, using 14% Tris-glycine precast gel (Novex, San Diego, CA). Coomassie-stained protein bands were scanned and analyzed using a densitometer (Duoscan T1200, Bio-Rad, Hercules, CA).
The purification of recombinant proteins was accomplished using metal affinity chromatography. The corresponding recombinant gene was cloned into the pET28a (Novagen) plasmid vector as mentioned previously, and expressed so that the soluble recombinant protein was synthesized as a fusion protein containing N-terminus six histidine residues. The resulting recombinant fusion protein with polyhistidine tag has a high affinity for ProBond resin (Invitrogen). Recombinant protein was purified through a Ni 2+ column with ProBond resin according to the following procedures. The cell-free supernatant containing 1-2 mg of polyhistidinetagged recombinant protein was loaded onto ProBond resin (Ni +2 ) column. Prior to sample loading, the resin was washed twice with 10 column volumes of binding buffer (50 mM potassium phosphate, 300 mM KCl and 20 mM imidazole, pH 7.0). Binding buffer contains 20 mM imidazole to minimize non-specific binding of untagged protein contaminants, and binding was carried out in a batch mode at 4 C. Subsequently, the resin was washed twice with 5-8 ml Tris-HCl (10 mM Tris, pH 8.0) prior to thrombin digestion step. Thrombin solution 50 ml (1 U/ml, Amersham, Catalog no. 27-0846-01) was mixed with Tris buffer 4950 ml, and the resulting reaction buffer was added to the column. After overnight thrombin digestion (16 h) at room temperature (22-25 C), the reaction solution was collected and properly concentrated using microcon YM-10 (Millipore, Ireland) before SDS-PAGE analysis.
The enzyme activity of recombinant arginine deiminase (ADI) was assayed by colorimetric determination of the enzyme reaction product, citrulline. The reaction mixture (0.1 M potassium phosphate, pH 6.5, 10 mM L-arginine and 0.1 ml enzyme solution in a final volume of 1 ml) was incubated at 37 C for 5 min, and the amount of citrulline was determined with diacetyl-monoxime (21). The sample heated to 95 C for 10 min and the optical density (A540 nm) was then determined. The protein concentration was determined using the Protein Assay Kit (Bio-Rad) with BSA as a standard. One unit of ADI is the amount of enzyme catalyzing 1 mmol of arginine to citrulline per min at 37 C under the assay conditions.

Peptide synthesis and in vitro DnaK-binding assay
Organic synthesis and purification of two peptides (GRIFLQD and GRALFQD) were implemented by Peptron, Inc. (Taejon, Korea). Each peptide was dissolved in Tris buffer (40 mM Tris-HCl and 0.5 mM ATP, pH 7.8), and the peptide concentration in the solution was adjusted to 10 mg/l. An aliquot of 20 ml of each peptide solution was dropped on PVDF membrane that was already wetted by methanol, and the blotted PVDF membrane was completely dried. [Through Ponceu S staining (0.25% Ponceu S in 1% acetic acid solution), we confirmed that a comparable amount of each peptide or protein was stably immobilized on the PVDF membrane.] The dried PVDF membrane was put into skim milk solution and slowly stirred for 30 min. Subsequently, the PVDF membrane was transferred into Tris buffer solution (40 mM Tris-HCl and 0.5 mM ATP, pH 7.8) containing 100 nmol of DnaK (Stressgen). After 1 h, the membrane was washed twice with phosphate-buffered saline buffer, followed by immunoblotting assay using anti-DnaK Ab (monoclonal IgG, mouse, Stressgen) and anti-mouse immunoglobulin G-horseradish peroxidase as primary and secondary antibodies, respectively.

Transmission electron microscopy (TEM)
Unstained samples of a purified protein solution were prepared for electron microscopy by air-drying small drops of a sample solution onto carbon-coated copper electron microscopy grids. To obtain stained images of protein nanoparticles, the electron microscopy grids containing air-dried samples were incubated with a 2% (w/v) aqueous uranyl acetate solution for 10 min at room temperature and washed 3-4 times with distilled water. Protein nanoparticle images were examined using the Philips Technai 120 kV electron microscope. Electron diffraction patterns were recorded from a selected area that is well occupied with protein nanoparticles in order to obtain high diffraction intensities. Particle size distributions were made by measuring 50 protein nanoparticles.

Solubility enhancer vector system using gene fusion of FTN-H
In a previous study, recombinant human ferritin H-, L-chain, and H-L hybrid were produced using the T7 expression system of E.coli (9). Interestingly, the recombinant human ferritin H-chain (FTN-H) and H-L hybrid were intracellular soluble and active proteins. However, despite high-sequence homology with FTN-H, recombinant human ferritin L-chain (FTN-L) was aggregated into inclusion bodies in E.coli cytoplasm (9,22). With the expectation that the N-terminus fusion of FTN-H could be a plausible solution for soluble recombinant protein synthesis in E.coli, we attempted to heterologously express the following proteins, all of which were produced as inclusion bodies upon direct expression in E.coli: EGF, hIL-2, human ppGRN, deletion mutants of human GAD (GAD 512-585 ), Mycoplasma ADI (ADI 132-410 ) and human mini-proinsulin (mp-INS) (23). was significantly enhanced. For instance, almost 80% of synthesized FTN-H::ppGRN was soluble in E.coli cytoplasm, although most ppGRN formed inclusion bodies even by GST fusion (data not shown). ADI catalyzes the hydrolysis of arginine into citrulline and ammonia, which have been reported to inhibit cell proliferation by arresting cells in the G 1 phase and also to demonstrate anti-cancer activity. As shown in Figures 1 and 2A, we confirmed that $30% of the expressed fusion protein, FTN-H::ADI 132-410 , was soluble in bacterial cytoplasm and biologically active as well, clearly showing arginine-degrading activity.
For the purification of ppGRN, FTN-H::ppGRN in a cellfree supernatant from whole-cell lysate was loaded to an Ni +2 column and retained inside the column due to the affinity interaction between Ni +2 and the N-terminus polyhistidine tag of FTN-H::ppGRN. Thrombin was subsequently added to the column to separate it from ppGRN by cleaving the fusion construct. As shown in Figure 2B Figure 3A). 2-HEDS is the oxidized form of DTT. When 2-HEDS is present in bacterial cytoplasm, its reduction is subject to the inducement of thiol oxidation of proteins, thereby promoting the formation of mis-linked disulfide bonds and protein aggregation. Comparing the results of reducing and non-reducing SDS-PAGE analyses, the aggregation of recombinant FTN-H by 2-HEDS seems to have occurred via intramolecular mislinkage of disulfide bridges (data not shown). From Figure 3A, even in the presence of 2-HEDS, the solubility of recombinant FTN-H was significantly increased when the HSP70 molecular chaperone, DnaK, was constitutively co-expressed. The co-expression of DnaK was carried out using a constitutive promoter localized in another expression vector, pHCE19 (2) (Bioleaders Inc., Korea) (20). Thus, it was possible to maintain the high cytoplasmic concentration of DnaK before the T7 expression of FTN-H was induced by IPTG. As shown in Figure 3B, the insoluble nature of recombinant FTN-L, however, never changed even with DnaK co-expression using the same constitutive promoter system. The results shown in Figure 3 suggest that FTN-H must be capable of having a significant interaction with DnaK, presumably via special amino acid residues acting as high-affinity motif(s) for DnaK. DnaK generally recognizes and binds to the hydrophobic region of the unfolded polypeptide chain. Thus, to identify the DnaK-binding motif(s), if any, the five hydrophobic clusters consisting of three amino acids (34VYL36, 81IFL83, 105LHL107, 133FIE35 and 161APE163) within the FTN-H sequence were first considered. They showed a relatively high hydrophobicity based on their theoretical hydropathy profile ( Figure 4A). Using the site-directed mutagenesis technique, each of the five hydrophobic clusters was mutated one at a time by replacing the hydrophobic amino acids in each cluster with arginine residue (34VYL36!34RRR36, 81IFL83!81RRR83, 105LHL107!105RHR107, 133FIE135! 133RRE135 and 161APE163!161RRE163), thereby resulting in the construction of five FTN-H-derived mutants. Each mutant was expressed in the E.coli T7 expression system, and its intracellular solubility was analyzed. The purpose of mutating the wild-type sequence was to identify the hydrophobic patch that is recognized and bound by DnaK. If a certain hydrophobic cluster is responsible for DnaK binding, replacing the hydrophobic amino acids comprising the hydrophobic patch with strongly charged amino acids, such as arginine, will significantly affect the solubility of the polypeptide chain in bacterial cytoplasm. After gene expression, three  FTN-H mutants (34RRR36, 81RRR83 and 105RHR107) were aggregated into inclusion bodies in E.coli cytoplasm ( Figure 4B), implying that the three hydrophobic tripeptides (34VYL36, 81IFL83 and 105LHL107) could be putative motifs interacting with DnaK. Although DnaK was constitutively co-expressed with the three mutants (34RRR36, 81RRR83 and 105RHR107) using the same constitutive promoter system, the inclusion body formation of the three FTN-H mutants was never prevented ( Figure 5). This is contrary to the case of wild-type FTN-H, in which the soluble FTN-H synthesis was resumed by DnaK co-expression even in the presence of 2-HEDS ( Figure 3A). Therefore, it seems that there must be a special motif(s) in FTN-H with a high affinity to DnaK, leading to the syntheses of soluble FTN-H and other soluble heterologous proteins with FTN-H fusion.

Identification and validation of a specific DnaK-binding motif
Case of the ferritin-light chain mutant. The alignment of the amino acid sequences between FTN-H and FTN-L shows that the sequence of a putative DnaK-binding motif, 81IFL83, is completely different from the aligned cognate sequence (77ALF79) of FTN-L ( Figure 6A). Through site-directed mutagenesis, the nucleotide sequence encoding 77ALF79 of FTN-L was replaced with the sequence coding for 77IFL79 so that FTN-L might have the putative DnaK-binding motif of FTN-H. The resulting substitution mutant of FTN-L (77IFL79) was expressed in E.coli. Surprisingly, the results of SDS-PAGE analysis showed that >50% of the synthesized FTN-L mutant was soluble in the bacterial cytoplasm ( Figure 6B), whereas almost 100% of wild-type FTN-L was aggregated into inclusion bodies (Figures 1 and 3). Figure 6C shows that it is very similar to the case of FTN-H, in which most of the FTN-L mutant was aggregated by the addition of 2-HEDS, but the synthesis of the soluble FTN-L mutant was resumed by DnaK co-expression ( Figure 6C). Consequently, it is evident that the tripeptide motif (IFL) from FTN-H played a critical role in increasing the solubility of the FTN-L mutant, probably by attracting the DnaK chaperone.
Case of the (GST mutant)::ppGRN. As described earlier in this study (Figure 1), the GST fusion protein, GST::ppGRN, was almost 100% aggregated upon expression in E.coli. The fusion expression of ppGRN was attempted using a GST mutant containing a DnaK-binding motif from FTN-H. First, several hydrophobic clusters in the N-terminus region of native GST were mutated by replacing the hydrophobic amino acids with arginine residues, and the cytoplasmic solubility of the resulting GST mutants was analyzed after their expression in the same recombinant E.coli system. As a result, a high degree of aggregation was observed in the synthesis of a GST mutant (85LAP87!85RRR87) ( Figure 7A). This is very similar to the previous results obtained with FTN-H mutants ( Figure 4). Thus, the hydrophobic cluster (85LAP87) and the sequence around 85LAP87 seem to be important sites in the interaction between GST and DnaK. To prepare GST mutants containing the putative DnaK-binding motif (81IFL83) of FTN-H, we first tried to replace the 85LAP87 of GST with the 81IFL83 of FTN-H, thereby constructing a substitution mutant of GST, 85LAP87!85IFL87 (GST-m1). We also constructed the following additional mutants of GST containing 80RIFLQ84 and 79GRIFLQD85 of FTN-H: 84LLAPV88!84RIFLQ88 (GST-m2) and 83QLLAPVN89!83GRIFLQD89 (GST-m3). Then, three fusion proteins of ppGRN (GST-m1::ppGRN, GST-m2::ppGRN and GST-m3::ppGRN) were synthesized using the same T7 gene expression system. From the results of the SDS-PAGE analysis, it is surprising that $50% of the synthesized GST-m3::ppGRN was in the form of soluble protein in E.coli cytoplasm ( Figure 7B). Figure 7C shows that almost 100% re-aggregation of GST-m3::ppGRN was also observed in the presence of 2-HEDS, but that the soluble GST-m3::ppGRN appeared again after DnaK co-expression, which is very similar to the cases of FTN-H and the FTN-L mutant.
Apparently, the two mutants, GST-m3 and FTN-L (77IFL79), have a common foreign sequence, GRIFLQD, originating from FTN-H, which is therefore likely to be a peptide motif with a high affinity to DnaK. It is generally accepted that DnaK binds to the hydrophobic region in an almost extended polypeptide chain of unfolded protein at the initial stage of protein folding. Therefore, the important factor determining the DnaK binding may not be protein conformation but the sequence structure of the binding motif. The sequence, GRIFLQD, consists of hydrophilic (polar) amino acid residues flanking the hydrophobic tripeptide. In the middle of the tripeptide, an amino acid with an aromatic side chain (Phe) is located (Figure 8). Presumably, such a unique sequence in the extended polypeptide chain can be easily recognized by DnaK.
In vitro DnaK-binding assay. Through an in vitro assay experiment, we investigated the actual binding of DnaK to the specific peptide GRIFLQD (named 'peptide A') above. In addition to the peptide A, we prepared another peptide, GRALFQD (named 'peptide B'), which exactly corresponds to the 79th-85th amino acids of FTN-L that were replaced by peptide A upon the preparation of the FTN-L mutant ( Figure 6B). From the results of Ponceau S staining ( Figure 9A), it seems obvious that both peptides A and B were well immobilized on the PVDF membrane. The addition of DnaK to the immobilized peptides and the subsequent immunoblotting analysis using anti-DnaK monoclonal Ab showed that DnaK bound only to peptide A ( Figure 9A), which clearly shows that the sequence 83GRILFQD89 within FTN-H has a high binding affinity for DnaK. We also implemented an additional in vitro assay of DnaK binding to FTN-H and the FTN-L mutant containing GRIFLQD, and to wild type FTN-L that did not contain GRIFLQD (negative control). Since DnaK binds to an unfolded nascent chain at the early stage of folding, we performed the overlay assay using purified and unfolded FTN-H, FTN-L and FTN-L mutant. (The purified proteins were unfolded using a denaturation buffer containing 1 mM DTT and 8 M urea.) Evidently, from Figure 9B, DnaK bound to only the proteins containing GRIFLQD, i.e. FTN-H and the FTN-L mutant, whereas DnaK binding never happened to wild-type FTN-L. Through this additional in vitro assay, we confirmed that the peptide motif GRILFQD (i.e. the 83rd-89th amino acids of FTN-H) has a high binding affinity to DnaK and seems to be directly related to the synthesis of soluble heterologous proteins. formed spherical nanoparticles with diameters of 10-15 nm, i.e. non-covalently cross-linked supra-molecules by selfassembly. The particle size seems to be quite uniform for each fusion protein. Considering that all the heterologous proteins (EGF, IL-2, ppGRN, GAD 512-585 and mpINS) were aggregated into inclusion bodies when directly expressed in E.coli, the results imply that the supra-molecule formation in the shape of a spherical shell may provide significant advantage(s) in preventing protein aggregation. It seems that upon supra-molecule formation, there must be a limitation in the increase of the supra-molecule size due to the assembly properties of FTN-H. Therefore, self-assembled supra-molecules were produced with a constant size of 10-15 nm, thereby avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins.
Immunoblotting analysis (western blot) following SDS-PAGE analysis of the purified FTN-H::ppGRN showed an apparent immunoreactive band ( Figure 10B). When western blot was carried out after native PAGE, however, no immunoreactive bands were visualized ( Figure 10C), which clearly indicates that a part of ppGRN, recognized by anti-ppGRN monoclonal Ab, was localized inside the supra-molecules. Thus, another plausible advantage of the self-assembled supra-molecule is its prevention of protein aggregation by non-specific hydrophobic interaction through the localization of critical hydrophobic patches of the target protein inside the supra-molecule.

DISCUSSION
As newly translated polypeptides emerge from the ribosome, they face a formidable task in the crowded environment of the cell: folding into their correct structure for proper functioning. Molecular chaperones are essential for the correct folding of a significant fraction of cellular proteins under both physiological and stress conditions (24), acting in a complex network as a main defense against protein aggregation. Some chaperones exhibit a holding activity, preventing polypeptides from aggregation (25)(26)(27). Others show a complementary folding role to assist in refolding and solubilization from aggregates (28,29). Chaperones also minimize aggregation by mediating the degradation of proteins that cannot be properly folded (30). The specific contribution of particular chaperones to this multifunctional folding network has only been partially identified. The Hsp70 chaperone DnaK serves as a central element of the chaperone network, assisting in de novo protein folding, protein translocation, oligomer dissociation and prevention of stress-induced protein aggregation (31)(32)(33). DnaK mediates protein folding through ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins (34).
The  (Figure 1). As explained above, DnaK plays a central role in the initial folding of the nascent polypeptide chain, and thus it seems reasonable to assume that FTN-H has a significant interaction with DnaK and contains specific motif(s) that DnaK efficiently recognizes and binds to. The results shown in Figures 3 and 9 strongly suggest that this assumption is not irrational. Recently, Vandenbroeck et al. (17) identified conserved DnaK-binding sites in the N-and C-terminal halves of helix B and C of diverse 4-helix-bundle proteins, respectively, that belong to the superfamily of interferon-g/interleukin-10-related cytokines. In this study, we also attempted to identify the specific amino acid residues of FTN-H, if any, with a high affinity to DnaK. Through systematic investigation using site-directed mutagenesis, the oxidative and forced protein aggregation technique and the DnaK coexpression vector system (Figure 4), several putative binding motifs were selected. It has been finally confirmed through an in vitro binding assay that DnaK recognizes an extended peptide strand composed of a hydrophobic core of three residues (Ile, Phe and Leu) and two flanking regions enriched in polar residues (Gly, Arg, Gln and Arg), such as the sequence GRIFLQD (Figure 8), which seems similar to the findings of Rudiger et al. (35). They reported that the DnaK-binding motif consists of a hydrophobic core of four or five residues enriched particularly not only in Leu, but also in Ile, Val, Phe and Tyr and two flanking regions enriched in basic residues. Although recombinant FTN-L was all aggregated into inclusion bodies in E.coli cytoplasm, the cytoplasmic solubility of the recombinant FTN-L mutant containing the putative DnaK-binding motif (GRIFLQD) was significantly increased ( Figure 6B). The efficacy of the binding motif was also validated using a GST mutant (GST-m3) containing the same motif sequence, i.e. the fusion protein GST-m3::ppGRN was expressed as a cytoplasmic soluble protein. The sequence  Figure 10A). Recently, Louzada et al. (36) proposed the mechanism of a quaternary structure (dimer) formation in the folding pathway of glutathione reductase (GR). They proposed that the assembly of the native GR dimer proceed via the initial formation of molten-globule monomers that dimerize to form a partially folded, expanded dimer. Structural rearrangements then take place at the dimer interface, leading to a final, compact dimer structure. Based on this proposed mechanism, it is presumed that the intermolecular association of ferritin (or ferritinfusion) monomers to form homopolymeric supra-molecules happens at the level of the partially folded (molten-globule) intermediate, and not at the level of the completely folded and thermodynamically optimal polypeptide chain. Protein aggregation, i.e. the off-pathway aggregation step, often initiates from partially folded intermediates upon intracellular chain folding and association (37). Within cells, nascent chains form sequentially on the ribosome, partially fold, and sometimes fail to reach their native state because of the intervention of various factors in a heterologous environment, including non-specific hydrophobic interactions with the intracellular cytoplasmic proteins (38), which sometimes cause the formation of large and insoluble protein aggregates, i.e. inclusion bodies. Supposing that as assumed earlier, the supramolecules of FTN-H or its fusion mutants are formed through the self-association of partially folded intermediates, they may have a distinct advantage in preventing the formation of insoluble protein aggregates; i.e. upon supra-molecule formation, the supra-molecule size would be limited by the assembly properties of FTN-H, and therefore, self-assembled supramolecules with constant sizes (10-15 nm) could be produced, thereby avoiding the undesirable formation of insoluble macro-aggregates of recombinant proteins ( Figure 11). As shown in Figure 11, a part of a target protein can be localized within the spherical shell of the supra-molecule upon the intermolecular association of a partially folded ferritin monomer, and thus, a protective environment against undesirable protein aggregation can be created. Evidently, from the results of the native PAGE and western blot analysis for FTN-H::ppGRN ( Figure 10B and C), an anti-ppGRN mAb-specific epitope was shown to be localized inside the supra-molecule. Hydrophobic patches of ppGRN may be hidden together inside, which, otherwise, may be involved in a non-specific hydrophobic interaction with the adjacent polypeptide chains, leading to protein aggregation.
In conclusion, this study proposes the dual function of FTN-H used for the fusion expression of heterologous proteins: (i) high-affinity interaction and association with DnaK and (ii) formation of self-assembled supra-molecules with limited and constant sizes, thereby preventing the undesirable formation of insoluble macro-aggregates of heterologous proteins and possibly creating a protective environment against protein aggregation. Similarly, Hidaka et al. (18) found that the propeptide of guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, has a dual function in the proper folding of the mature peptide. Thus, the N-terminus propeptide of pro-GCAP-II is critical not only for disulfide-coupled folding but also for the net stabilization (or dimerization) of pro-GCAP-II. The ferritin supra-molecular structure at the nanometer scale (or spherical protein nanoparticles) can contribute to the enhancement of the stability of synthesized recombinant proteins and can also be used as a potent 3D probe structure for an ultra-sensitive protein chip if a special expression system is designed so that the captured probe peptide can be properly displayed at the surface of the ferritin nanoparticles.