ABSTRACT
Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.
Nearly all patients infected with hepatitis C virus (HCV) develop a persistent infection that can progress to cirrhosis, hepatocellular carcinoma, and liver failure. Limited therapy is available, and a large number of viral isolates are resistant. HCV is able to avoid host defenses in part due to the high variability among different strains and isolates. The development of better therapies has also been hindered by the lack of an established in vitro cell culture system and a useful laboratory animal model.
HCV has a positive-strand RNA genome about 9,600 bases long (12). The most conserved and highly structured portion of the HCV genome includes the 5′ untranslated region (5′ UTR) and about 30 bases of coding sequence (14, 15, 32) which have been shown to contain an internal ribosome entry site (IRES) (6, 14). Despite the lack of a 5′-terminal cap, protein synthesis still initiates, although at an AUG codon downstream from several others. IRESs have been found in both viral genomes and cellular mRNAs (15, 24, 26), yet they fail to share significant primary sequence homology. These IRESs range in size from 300 to 1,500 nucleotides and possess both secondary and tertiary structural elements. Since there is no extensive homology among IRES domains from viral or cellular mRNAs, higher-order structure along with limited and widely separated primary sequence elements may combine to create IRES ribosome recognition sites (13, 14).
Beginning with work by Steitz (31) and others (9, 28), the isolation and sequence analysis of ribosome binding sites (RBSs) from radioactive polycistronic mRNAs of prokaryotes have been used to define the mRNA sequences primarily responsible for bringing a ribosome into the correct orientation for translation (25). An RBS is the ribosome-protected region of an mRNA under conditions which would otherwise lead to complete mRNA solubilization. RBSs remain attached to the ribosome and, thus, are separated from the bulk of the mRNA molecule by sedimentation after exhaustive digestion of initiation complexes with RNase (usually pancreatic RNase A) (23, 25). A similar approach was used to isolate RBSs from capped monocistronic mRNAs of eukaryotes (18-22).
Studies on HCV IRES sequences and ribosomal subunits have so far concentrated on 40S preinitiation complexes (17, 27). No direct characterization of the 80S RBSs from HCV, or any IRES-containing mRNA, has yet been reported. We report our discovery that a series of discontinuous sequences combine to form a ribosome binding domain many times the conventional size, and we compare these findings to 48S preinitiation complexes.
MATERIALS AND METHODS
In vitro transcription.The plasmid pN(1–4728) containing the first 4.7 kb of HCV sequence adjacent to the phage T7 promoter was a gift from Stanley Lemon, University of Texas, Galveston. The plasmid was cleaved by three different restriction enzymes: AatII,SacII, and BamHI. When these templates were transcribed in vitro, three 32P-labeled RNAs spanning bases 1 to 402, 1 to 645, and 1 to 1349 were produced. The plasmid pβHb containing rabbit β-globin cDNA was a gift from Karen Browning, University of Texas, Austin. This plasmid was cleaved withHindIII to produce the proper template for transcription. The transcription reactions are based on earlier published work from this laboratory (2, 7), and specific activities of 4.13 × 106 or 8.25 × 107 dpm/μg were generally used. [α-32P]GTP (NEN Life Science Products, Boston, Mass.) was used in all labeled transcriptions unless noted otherwise.
Ribosome binding.Ribosome binding reactions followed the standard translation protocol of Promega (Madison, Wis.). Each sample contained 35 μl of nuclease-treated rabbit reticulocyte lysate (RRL) (Promega), 1 μl of amino acid mixture (Promega), and enough water so that the final reaction volume equaled 50 μl. If necessary, 10 mM EDTA was added to control reactions at this time (34). Each reaction mixture was incubated at 30°C, and 100 μM anisomycin or sparsomycin was added after 1 min. Edeine (1 μM) or aurin tricarboxylic acid (100 μM) was added after 4.5 min of incubation. Edeine was a gift from Z. Kurylo-Borowska. After 5 min at 30°C, 1 μg of radiolabeled mRNA was added. The samples were incubated for another 10 min (15-min total incubation) and then immediately placed on ice. Pancreatic RNase A was added to each sample (25.6 μg/ml [final concentration] for HCV RNAs and 9.6 μg/ml for β-globin RNA) followed by incubation on ice for 15 min.
Sucrose density gradient analysis.After 15 min on ice, 250 μl of gradient buffer (25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl [pH 7.5], and 1 mM dithiothreitol) was added to each reaction and the samples were loaded onto 5-ml 15 to 30% sucrose gradients using the same gradient buffer. Gradient buffer similar to that described above, but without MgCl2 and containing 10 mM EDTA, was used for both dilution of samples and formation of 5-ml 15 to 30% sucrose gradients containing EDTA. All samples were then centrifuged for 2 h at 2°C and 45,000 rpm (189,378 × g average). Two-drop fractions were collected as described before (28, 31), and those corresponding to the appropriate peaks were pooled. The total volume containing the 80S or 48S peaks was approximately 1 ml. Protected RNA was recovered from pooled fractions as described before (20-22). Pooled 80S or 48S peaks were added to 1 ml of phenol containing 0.5 g of urea, 10 μl of mercaptoethanol, 10 μl of 10% sodium dodecyl sulfate, and 10 μl of 200 mM EDTA at ambient temperature. The mixture was vortexed, 1 ml of chloroform was added, and after more vortexing, the phases were separated by centrifugation. The aqueous layer was collected and ethanol precipitated.
RNA fingerprinting.RNA fingerprinting, a technique in which RNA molecules are digested with RNase T1(Calbiochem, San Diego, Calif.) and the digestion products are separated in two dimensions by charge and size, has been described previously (3).
RESULTS
80S ribosomal complex formation on HCV and β-globin RNAs.Internally labeled transcripts containing the complete HCV 5′ UTR and various lengths of HCV mRNA coding sequence were used to form 80S initiation complexes in RRL. Eukaryotic β-globin mRNA was used as a control since its RBS has already been characterized (21, 22). To interrupt the translation process at the point of first peptide bond formation, translation inhibitors, anisomycin and sparsomycin, were added to the reactions (33). At the concentrations used here, these inhibitors allow the 80S ribosomal complex to bind to the start codon but prevent translation elongation on capped mRNAs beyond the dipeptide state, thus permitting a stable 80S ribosomal-mRNA complex to form (20-22). The radiolabeled RNAs were incubated in the lysate in the presence of either anisomycin or sparsomycin, treated with RNase A to solubilize all parts of the mRNA not protected by ribosomes, and loaded on 15 to 30% sucrose gradients for centrifugation.
RNA sedimentation profiles were obtained for HCV and β-globin RNAs after collecting fractions and determining their radioactivity (Fig.1). When the RNAs are centrifuged alone, without any RNase treatment, they both are found in the top of the gradients (Fig. 1K and L). In the presence of anisomycin and after treatment with RNase, peaks corresponding to the 80S ribosomal initiation complex and protected radiolabeled RNA form and can be seen in the profiles of both HCV and β-globin RNAs (Fig. 1A and B). Similar results were seen when sparsomycin was added to the reaction (data not shown). These 80S ribosomal complexes cannot form when the RRL is pretreated with 10 mM EDTA (Fig. 1C and D). Similarly, these complexes dissociate when centrifuged in sucrose density gradients containing 10 mM EDTA (Fig. 1E and F). When fractions from 80S regions obtained with and without EDTA were quantified from a scaled-up preparation, the EDTA-treated samples had only 4 to 5% of the radioactivity found in those obtained with the complete system. Thus, recovery of the vast majority of the 80S-protected RNA fragments requires protein synthesis initiation.
HCV RNAs of 645 bases and β-globin RNAs of 602 bases bind to 80S ribosomal initiation complexes. (A and B) Sucrose gradient sedimentation profiles of radiolabeled 645-base HCV or 602-base β-globin RNAs incubated in RRL containing anisomycin, treated with RNase, and centrifuged in sucrose density gradients containing 1 mM MgCl2. (C and D) Sedimentation profiles of radiolabeled HCV or β-globin RNAs incubated in RRL containing anisomycin and 10 mM EDTA, treated with RNase, and centrifuged in sucrose density gradients containing 1 mM MgCl2. (E and F) Sedimentation profiles of radiolabeled HCV or β-globin RNAs incubated in RRL containing anisomycin, treated with RNase, and centrifuged in sucrose density gradients containing 10 mM EDTA. (G and H) Sedimentation profiles of radiolabeled HCV or β-globin RNAs incubated in RRL containing anisomycin and ATA, treated with RNase, and centrifuged in sucrose density gradients containing 1 mM MgCl2. (I and J) Sedimentation profiles of radiolabeled HCV or β-globin RNAs incubated in RRL containing edeine, treated with RNase, and centrifuged in sucrose density gradients containing 1 mM MgCl2. (K and L) Sedimentation profiles of radiolabeled HCV or β-globin RNAs incubated in RRL and centrifuged in sucrose density gradients containing 1 mM MgCl2. Both the HCV and β-globin RNAs are of approximately equal lengths, about 600 bases. All profiles show the counts per minute plotted against the fraction number of the gradient. The first fraction is taken from the bottom of the gradient, and the last is taken from the top. The 80S and 48S ribosomal peaks are indicated.
Interestingly, the HCV profile of the RNA incubated in RRL containing anisomycin (Fig. 1A) or sparsomycin (data not shown) also contains a peak corresponding to a 40S subunit-mRNA complex (fractions 18 to 22) not observed with β-globin mRNA (Fig. 1B) (21, 22). Furthermore, addition of the translation inhibitor aurin tricarboxylic acid, which has been shown to block all binding of globin mRNA to rabbit reticulocyte ribosomes (20-22, 33), abolishes both globin and HCV 80S ribosome-protected peaks but allows the HCV 40S peak to persist (Fig. 1G and H). In the presence of edeine, which allows 48S complexes to accumulate but prevents 60S subunit association, similar 48S peaks are observed for both HCV and β-globin mRNAs (Fig. 1I and J).
Analysis of HCV and β-globin protected RNA fragments.The fractions containing the 80S ribosome peaks were pooled, and the radioactive, protected RNA comprising the RBSs was isolated (20-22). These RNAs were then electrophoresed on a denaturing polyacrylamide gel (Fig. 2). The β-globin RNA protected by the 80S ribosomal initiation complex (Fig. 1B) is four bands approximately 40 to 35 bases in length (Fig. 2, left lane). In contrast, the protected HCV RNA is up to 30 bands varying in length from more than 100 bases to approximately 9 bases. It is evident that many more bases of sequence are protected in the HCV IRES than in the β-globin mRNA. By identifying these IRES sequences, we will have the first direct indication of how the IRES specifies ribosome binding.
The 80S ribosomal initiation complex protects many fragments of HCV RNA of various lengths. The protected RNA fragments from the 80S peaks of both β-globin (left lane) and HCV (1 to 1349) (right lane) mRNAs were isolated, electrophoresed on a 15% denaturing polyacrylamide gel, and subjected to autoradiography. The 80S ribosomal complexes accumulated after treatment with sparsomycin. O, origin; XC, xylene cyanol; Bφ, bromophenol.
The protected RNA fragments were then subjected to RNA fingerprinting analysis (3). These β-globin mRNA fragments and the control intact β-globin mRNA were fingerprinted (Fig.3A and B). The complex fingerprint pattern of the intact control (Fig. 3A) is greatly simplified for the ribosome-protected RNA (Fig. 3B), signifying that much of the 600-base RNA was not protected and is missing from the pattern. The spots from the 80S-protected β-globin mRNA fingerprint were eluted, and secondary RNase digestions were performed to determine the sequence of each RNase T1-resistant oligonucleotide. The presence of the previously reported 80S ribosome-protected sequence (21, 22) was thus confirmed and found to contain bases 43 to 82 of rabbit β-globin mRNA, a sequence 40 bases in length (see Fig. 5A). The shorter bands seen in the left lane of Fig. 2 contain this same sequence minus a few bases from either end, a “frayed” end. The shortest protected band is defined as the “core” sequence. The intact HCV mRNA (Fig. 3C) and the 80S ribosome-protected fragments (Fig. 3D) were also fingerprinted. A simpler pattern is seen in the 80S ribosome-protected HCV fingerprint.
The RNA fingerprints of the 80S ribosomal complex-protected β-globin and HCV mRNAs are much simpler. (A) Intact β-globin mRNA was subjected to RNA fingerprinting. The radiolabeled RNA was subjected to exhaustive RNase T1 digestion. The resulting oligonucleotides then were separated in the first dimension by charge (from right to left) and in the second dimension by size (from bottom to top). (B) The protected β-globin RNA fragments were isolated from the 80S ribosomal initiation complex peak (like that of Fig. 1B) and subjected to RNA fingerprinting. The 80S ribosome-protected mRNA produces a simpler pattern than does the intact control mRNA. (C) Control HCV mRNA containing bases 1 to 1349 was fingerprinted. (D) HCV mRNA (bases 1 to 1349) from the 80S ribosomal peak (like that of Fig. 1A) was fingerprinted. This fingerprint pattern is much simpler than the control shown in panel C.
The 80S-protected fragments of HCV mRNA are discontinuous and noncontiguous. The HCV RNA genome is depicted here as a single thick black line. The recovered, protected fragments are depicted above the genome as thin black lines. All protected fragments lie within bases 124 to 392. No protected fragments were found between bases 191 and 202.
The HCV RBS contains a series of discontinuous fragments.The gel pattern for the HCV RBS (Fig. 2, right lane) is reproducible, and each band was analyzed in detail. On two occasions, separate HCV or β-globin mRNA transcripts labeled with [α-32P]GTP or [α-32P]CTP were analyzed. In the case of the HCV RBS, at least 12 gel band sequences (Fig. 2, right lane) were characterized in each of the four preparations. Each of the individual gel bands of HCV protected RNA was eluted and fingerprinted (data not shown). Using secondary RNase digestions of the RNase T1-resistant oligonucleotides of each fingerprint (1, 3), the sequence of each purified 80S ribosome-protected fragment was determined. The termini of these sequences identified points of RNase cleavage within, or at the borders of, HCV RBS elements. The longest protected sequence contains 128 bases, and the shortest contains 9 bases. All of the protected fragments fall between bases 124 and 392 of the HCV map (Fig. 4). The largest fragment covers bases 204 to 331, which contain the right side of stem-loop III and much of the pseudoknot region. Another large fragment contains the left side of stem-loop III from bases 124 to 191. Another fragment contains the start codon and 36 bases of downstream coding sequence. The sequences recovered four times or more from the HCV IRES, either alone or as part of a larger fragment, are defined here as core sequences. These protected fragments must interact to form a specific ribosome binding domain.
The RBS of the approximately 600-base β-globin mRNA is 40 bases in length (blue box) with the start codon located at bases 56 to 58 (Fig.5A). The core sequence (orange box), the smallest protected fragment, is 35 bases in length. In contrast, the RBS of the HCV genomic mRNA, drawn to a similar scale, comprises two segments 68 and 189 bases long, respectively (blue boxes), separated by a 12-base region of unprotected RNA. The start codon is located at bases 342 to 344. The core elements of the HCV RBS, the most frequently protected sequences, consist of five noncontiguous RNA segments (orange boxes) which contain a total of 108 bases, as follows: 128 to 138 (Fig. 5A, domain 1), 145 to 156 (Fig. 5A, domain 2), 237 to 249 (Fig. 5A, domain 3), 286 to 324 (Fig. 5A, domain 4), and 342 to 373 (Fig. 5A, domain 5). Each core is substantially or completely protected from RNase digestion and was recovered both alone and covalently linked to additional RNA segments (Fig. 4). Two kinds of noncore elements are recovered only as part of larger segments containing at least one core element. Noncore elements are sometimes cleaved by RNase A during RBS isolation, although much less frequently than the rest of the HCV mRNA. One subset of HCV noncore elements has not been observed to undergo cleavage. The three segments comprising this group are indicated by pairs of vertical arrows connected by horizontal dashed lines (Fig. 5A).
The RBS of HCV mRNA is large and discontinuous. (A) The RBSs of β-globin and HCV mRNAs are shown as blue boxes. The core regions are represented by orange boxes. HCV mRNA has five core regions which are discontinuous. The three segments comprising noncore elements which have not been observed to undergo cleavage are indicated by vertical arrows connected by horizontal dashed lines. (B) The HCV RBS domains are mapped on the secondary structure of the HCV IRES. The protected regions are shown in blue, and the core sequences are shown in orange.
The protected regions of the HCV IRES are shown in blue, and the core sequences are shown in orange (Fig. 5B). Core domain 1+4 contains the pseudoknot of the HCV IRES, core domain 2+3 is located in the base-paired region below stem-loops IIIa and IIIc, and core domain 5 contains the initiator AUG codon and is the most similar to the globin mRNA core domain. Arrows indicate termini of noncore elements lacking cleavage sites (Fig. 5).
As previously described in the literature (21), the edeine-induced 48S ribosomal complex protected a larger region of β-globin mRNA of approximately 60 bases in length (Fig.6, lanes 1 and 3). The 48S-protected HCV RNA fragments, like the 80S-protected RNA fragments, range in length from 10 bases to over 100 bases (Fig. 6, lane 4). These protected fragments were isolated and characterized in a manner similar to that described for the 80S-protected fragments (data not shown). The largest protected fragment lies between bases 259 and 355. Another large fragment covers bases 125 to 192. The 48S-protected region is very similar to that protected by the entire 80S ribosomal complex. However, in contrast to the RBS, the most 3′ protected base is nucleotide 355, which corresponds with the end of stem-loop IV. No other downstream nucleotides are protected. Otherwise, the 48S-protected region, like the RBS, contains stem-loop III minus the apical loop, the pseudoknot region, and stem-loop IV.
The 48S ribosomal initiation complex also protects many fragments of HCV RNA of various lengths. The protected fragments from the anisomycin-induced 80S peaks of both β-globin (lane 1) and HCV (bases 1 to 645) (lane 2) mRNAs were electrophoresed on a 15% denaturing polyacrylamide gel and subjected to autoradiography. The edeine-induced 48S ribosomal complexes of β-globin (lane 3) and HCV (bases 1 to 645) (lane 4) mRNAs are also shown. XC, xylene cyanol; Bφ, bromophenol.
DISCUSSION
We report here the initial results of our studies involving direct analysis of the interaction between mammalian ribosomes and the HCV IRES using the conventional capped β-globin mRNA for comparison. Under conditions of pancreatic RNase digestion which lead to complete solubilization of both HCV mRNA and the control globin mRNA species, we find, in both cases, specific protection of RBS domains by initiating 80S ribosomes. The globin mRNA RBS, under the translation-blocked conditions used here, is typical of a conventional capped mRNA (18-22), comprising a 40-base region with a 35-base core domain containing the AUG start codon (Fig. 5A). In contrast, the RBS sequences of the HCV IRES contain substantially more bases (257) than those of the globin mRNA RBS, including five core elements totaling 108 bases. Unlike globin RNA, in which the start codon is present in all RNase-protected fragments, many of the HCV IRES-protected fragments do not contain the initiator AUG codon.
It is clear that, even when we adopt the conventional secondary-structure folding pattern for the HCV IRES (Fig. 5B) (4, 10, 11), we see three separate core domains: 1+4, containing the pseudoknot structure; 2+3, a duplex stem between stem-loop IIId and stem-loops IIIb and IIIc; and 5, which contains the AUG start codon and the first 30 bases of coding sequence. These results suggest to us a minimum of three different sites on the 80S ribosome which protect substantial IRES core sequences while ensuring accurate initiation. In this regard, it is likely that core sequence 5 occupies the ribosomal mRNA binding groove with its AUG triplet precisely aligned with initiator tRNA in the P site. HCV IRES core domain 5 would thus occupy the position analogous to that of the RBS of a capped mRNA.
Which factors or ribosomal proteins are responsible for protection of such a large amount of the HCV IRES RNA? The HCV IRES has a distinctive mechanism for ribosomal complex formation (27) and, therefore, may require novel factors and/or a heretofore unknown order of assembly; in this sense, the HCV-80S ribosomal initiation complex may be unlike typical 80S ribosomal complexes with capped mRNAs and may contain unknown factors or factors normally not present after ribosomal complex initiation. Since our 80S ribosomal complexes do not include purified components and are formed in RRL, all cellular factors or proteins are available for the perhaps unique HCV-80S ribosomal initiation complex formation. In this light, perhaps the ribosomal entity responsible for protecting core domain 2+3 could be eukaryotic initiation factor 3 (eIF3). While this initiation factor is thought to exit the ribosome upon 60S subunit addition in the binding of conventional capped mRNAs (8), the HCV IRES shows a strong and direct binding reaction to purified eIF3 (5, 17, 27, 29). Therefore, an HCV IRES domain remaining bound to eIF3 could lead to a delayed exit of this factor from 80S ribosomes, resulting in the protection of core domain 2+3 observed here.
The protection from RNase of our core domain 1+4, which contains the HCV IRES pseudoknot region, leads us to postulate the involvement of a third ribosomal domain. We have found in collaboration with J. Gomez and colleagues of Barcelona, Spain, that the host pre-tRNA processing enzyme RNase P can cleave HCV RNA at two sites, one of which is located in the HCV IRES near the initiator AUG codon (A. Nadal, M. Martell, J. R. Lytle, H. D. Robertson, B. Cabot, J. I. Esteban, R. Esteban, J. Guardia, and J. Gomez, submitted for publication). Since RNase P, in the absence of external guide sequences, cleaves only tRNA-like structures, this result suggests that a tRNA-like structure exists within the HCV IRES at a site near our pseudoknot-containing core domain 1+4. Ribosomes bind tRNA at three sites: the A site, the P site, and the E site. We suggest that this tRNA-like domain of the HCV IRES, and perhaps analogous domains in other IRESs, could occupy one of the tRNA binding sites of the initiating 80S ribosome.
Our work here focuses on the HCV RNA protected by 80S ribosomal initiation complexes, while work done by others has determined locations of HCV RNA involved in 40S preinitiation complexes (with or without additional factors) (17, 27). Significantly, several of our domains have already been shown to be important regions for binding. Core domain 3 has been shown to be involved in eIF3 binding to the HCV IRES (27). Similarly, bases from our core domains 4 and 5 have been shown to interact with the 40S subunit alone using the indirect toeprinting assay to measure 40S subunits' ability to block primed DNA synthesis (27). Several laboratories have determined stem-loop IIId to be important for translation (16, 17), and we find these bases in a protected, but not core, domain. This region may be more important for an earlier initiation step in protein synthesis and not as strongly protected at the peptide formation stage.
The HCV RNA sequences protected by the 48S preinitiation complex (Fig.1I and J) are very similar to those protected by the 80S ribosomal complex. This result is similar to that described previously for β-globin mRNA: the 48S-protected region of β-globin mRNA contains the entire RBS sequence but also additional 5′ sequence from the 5′ UTR (21). Initially, the large amount of 48S-protected HCV RNA may seem surprising. However, the recent cryoelectron microscopy map made by Spahn et al. (30) of the HCV IRES complexed with the 40S ribosomal subunit shows that almost all of the HCV RNA binds to the 40S subunit on the side opposite where the 60S subunit associates. Only stem-loop II wraps around the 40S subunit to the 60S side, and this domain has so far not been shown to be protected from digestion by either the 48S or the 80S complex. This finding implies that the 60S subunit would not afford any additional protection from RNase and that the protection of the HCV IRES by the 48S preinitiation complex could indeed be very similar to that provided by the entire 80S ribosomal complex.
The HCV RBS is the largest RBS found to date. The generality of this HCV RBS paradigm for other cellular and viral IRESs remains to be seen. However, the discontinuous nature and considerable size of the HCV RBS are likely to be shared by related, viral IRESs. Confirmation of a pattern of common structural features will help explain the action and specificity of IRESs.
ACKNOWLEDGMENTS
This work was supported in part by grant U35-8010 from the New York State Science and Technology Foundation's Centers for Advanced Technology Program and by Public Health Service grant DK-56424 from NIH.
We thank S. Genus and P. Mercado for excellent technical assistance, E. Kanner for help in growing DNA plasmids, K. Browning and S. Lemon for gifts of plasmid DNA, Z. Kurylo-Borowska for the gift of edeine, and J. Gomez for helpful discussions.
FOOTNOTES
- Received 12 February 2001.
- Accepted 17 May 2001.
- Copyright © 2001 American Society for Microbiology