Rnasee and the Other Constituents of the RNA Degradosome Are Components of the Bacterial Cytoskeleton

RNaseE and the other constituents of the RNA degradosome are components of the bacterial cytoskeleton

Aziz Taghbalout and Lawrence Rothﬁeld*

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06032

Communicated by M. J. Osborn, University of Connecticut Health Center, Farmington, CT, November 27, 2006 (received for review July 21, 2006) RNaseE is the main component of the RNA degradosome of The degradosome includes at least three other proteins, RNA Escherichia coli, which plays an essential role in RNA processing and helicase B (RhlB), polynucleotide phosphorylase (PNPase), and decay. Localization studies showed that RNaseE and the other enolase (12–15). The RNA degradosome is required for the normal known degradosome components (RNA helicase B, polynucleotide maturation of transfer and ribosomal RNA and for degradation of phosphorylase, and enolase) are organized as helical filamentous most messenger RNAs (16–18). In degradosome-dependent structures that coil around the length of the cell. These resemble mRNA decay, RhlB facilitates the degradation of structured RNA, the helical structures formed by the MreB and MinD cytoskeletal and RNaseE provides the endoribonuclease activity that cuts the proteins. Formation of the RNaseE cytoskeletal-like structure re- RNA into fragments that are further degraded by the 3Ј35Ј quires an internal domain of the protein that does not include the exoribonuclease activity of PNPase (reviewed in ref. 19). The role domains required for any of its known interactions or the minimal of enolase in this process is unclear (20). Recently, enolase was domain required for endonuclease activity. We conclude that the proposed to play a regulatory role in the degradation of specific constituents of the RNA degradosome are components of the E. coli RNAs such as ptsG mRNA (21). cytoskeleton, either assembled as a primary cytoskeletal structure We report here that RNaseE and the other degradosome com- or secondarily associated with another underlying cytoskeletal ponents are all organized as helical filamentous structures that wind element. This suggests a previously unrecognized role for the MICROBIOLOGY bacterial cytoskeleton, providing a mechanism to compartmental- around the length of the cell. The structures resemble the helical ize proteins that act on cytoplasmic components, as exemplified by structures formed by the cytoskeletal proteins MinD and MreB, but the RNA processing and degradative activities of the degradosome, formation of the cytoskeletal-like RNaseE structures is indepen- to regulate their access to important cellular substrates. dent of MinD or MreB. The RNaseE domain responsible for its cytoskeletal organization is separate from the RNaseE domain that RNA processing ͉ PNPase ͉ enolase ͉ RNA helicase contains the essential endoribonuclease activity. The present results indicate that the RNA degradosome exists as a cytoskeletal struc- E. coli t is well established that the eukaryotic cytoskeleton includes ture in , thereby compartmentalizing RNA degradative and Istable structures that are composed mainly of intermediate processing activities within the cell. This type of compartmental- filament proteins and dynamic structures such as microtubules and ization could provide a general mechanism to spatially sequester actin filaments that can assemble and redistribute within the cell in proteins or protein complexes that act on cytoplasmic components response to specific cellular cues. It has recently been established and thereby regulate their access to specific substrates within the that a variety of cytoskeletal-like structures are also present in cytoplasm. bacterial cells (1). By analogy to the eukaryotic cytoskeleton, bacterial cytoskeletal elements are defined as filamentous struc- Results tures, each based on polymers of a single class of protein, organized Screening for MinD-Interacting Proteins. As part of a study to identify into long-range ordered structures within the cell. Among these proteins that interact with bacterial cytoskeletal elements, we used elements are proteins that form membrane-associated helical struc- the yeast two-hybrid system to screen an E. coli genomic library for tures that extend along the length of Escherichia coli cells, such as genomic fragments coding for proteins that interact with MinD. the actin bacterial homolog MreB (2, 3) and the uniquely bacterial Ten genomic clones that interacted with the MinD bait were cytoskeletal protein MinD (3). identified out of a total of 12.3 ϫ 106 yeast colonies. Six clones Bacterial cytoskeletal structures play a role in a number of cell contained DNA coding for part of the MinC protein, and one functions, including cell shape determination (4), division site contained MinD DNA. These are expected because MinD interacts selection (3), establishment of cell polarity (5, 6), and segregation with itself and with MinC (22, 23). The three other clones contained of chromosome and plasmid DNA (7, 8). To accomplish these chromosomal inserts corresponding to the central domain of the rne functions, the cytoskeletal structures can act as lattices for the gene, coding for the E. coli RNaseE protein. The three inserts assembly and localization of functional protein complexes. For started from the same position, His-378, but differed in the length example, the MreB helical cytoskeleton plays a role in cell shape of the RNaseE domains, which extended to Gln-659, Arg-679, and determination by directing the helical organization of murein cell Gln-724, respectively (Fig. 1). wall biosynthetic enzymes (9). Similarly, MinD helical cytoskeletal structures play a role in the proper mid-cell placement of the E. coli cell division site by serving as a scaffold for the dynamic localization Author contributions: A.T. and L.R. designed research; A.T. performed research; A.T. and of the MinC and MinE division site-selection proteins (reviewed in L.R. analyzed data; and A.T. and L.R. wrote the paper. ref. 10). The authors declare no conflict of interest. As part of a study to identify cytoskeleton-associated elements, Abbreviations: PNPase, polynucleotide phosphorylase; RhlB, RNA helicase B; Yfp, yellow we used the yeast two-hybrid system to screen an E. coli genomic fluorescent protein; AD, activation domain. library for proteins that interact with the MinD protein. This *To whom correspondence should be addressed. E-mail: [email protected]. identified RNaseE as a MinD-interacting protein. RNaseE is an This article contains supporting information online at www.pnas.org/cgi/content/full/ essential endoribonuclease of 1,061 aa (11) that acts as a scaffold for 0610491104/DC1. the assembly of a multiprotein complex, the RNA degradosome. © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610491104 PNAS ͉ January 30, 2007 ͉ vol. 104 ͉ no. 5 ͉ 1667–1672 Downloaded by guest on October 1, 2021 Fig. 1. Schematic representation of RNaseE and Yfp-labeled RNaseE constructs. RNase domains are depicted as described in ref. 41. S1 domain (S1 RNA-binding domain), RBD (arginine rich RNA-binding domain), RhlB (RhlB- binding domain), enolase (enolase-binding domain), and PNPase (PNPase- binding domain) are shown. The region that includes the endoribonuclease catalytic domain is indicated (26). The black rectangles represent the RNaseE fragments that interacted with MinD in the yeast two-hybrid screen. The Yfp-labeled RNaseE constructs are shown in gray.

RNaseE Is Organized as a Cytoskeletal Structure in Vivo. The yeast two-hybrid results suggested an interaction between RNaseE and MinD, which is organized as a helical, membrane-associated cytoskeletal structure within the cell. We therefore asked whether RNaseE showed a similar cellular organization, using RNaseE fused to yellow fluorescent protein (Yfp) to study its localization pattern in living E. coli cells. Yfp fused to either the N terminus or the C terminus of RNaseE did not interfere with the ability of the protein to correct the lethal phenotype of a ⌬rne mutant (data not shown). Fluorescence microscopy revealed that RNaseE-Yfp was organized as a double-helical filamentous structure that coiled around the cell periphery and extended between the two poles (Fig. 2B). Fig. 2. Cytoskeletal-like organization of the RNA degradosome compo- Similar results were obtained when RNaseE-Yfp was expressed nents. (A) Differential interference contrast micrographs of strain AT1/pRNE1 ⌬ ⌬ under control of P (Fig. 2B) or when rne::yfp was substituted for [ rne/Plac-rne::yfp]. (B) Strain AT1/pRNE1 [ rne/Plac-rne::yfp] cell showing lac coiled structure of RNaseE-Yfp. (C–K) Immunofluorescence micrographs using the native rne gene in the chromosome under control of the normal anti-HA antibody (C and E–K) or purified anti-RhlB antibody (D). (C) Strain rne promoters (data not shown). AT25 [rne::HA]. (D) Strain MC1000. (E) Strain AT18 [eno::HA]. (F) Strain AT19 Confirmation that the localization pattern of RNaseE-Yfp was [pnp::HA]. (G) Strain AT27 [rne1–659::HA]. (H and I) Strain AT28 [rne1–417::HA]. not an artifact due to the fusion to Yfp was obtained by study of rne1–417 cultures contain cells of variable length, including filamentous cells chromosomally encoded RNaseE tagged with the HA epitope (see Results). (J) A22-treated AT25 [rne::HA] (see Results for details). (K) Strain ⌬ Ϫ (RNaseE-HA). Immunofluorescence microscopy showed that the AT41 [ min, rne::HA]. Because of the Min phenotype, the cells are predom- inantly short filaments. (Scale bars: 1 ␮m.) RNaseE-HA was organized into coiled structures similar to those observed with RNaseE-Yfp (Fig. 2C). We conclude that RNaseE is organized as helical cytoskeletal-like structures in E. coli cells that RNaseE Organization Is Independent of MreB and MinD Cytoskeletal resemble the previously described membrane-associated helical Structures. MreB forms coiled cytoskeletal structures that can be structures formed by cytoskeletal proteins MinD and MreB (2, 3). disrupted by treatment with low concentrations of A22 [S-(3,4- dichlorobenzyl) isothiourea] without significant change in cell Localization of the Other RNA Degradosome Components. Within shape (8). To determine whether the MreB structure is required for the cell, RNaseE is associated in the RNA degradosome with the cytoskeletal-like organization of RNaseE, the localization pat- RhlB, PNPase, and enolase (24). Because Yfp labeling and immu- tern of RNaseE-HA was determined by immunofluorescence in nofluorescence studies showed that RNaseE is organized as a cells in which the MreB helical structure was disrupted by A22 cytoskeletal-like structure, we next asked whether the other RNA treatment. As expected from other reports (25), when exposed to degradosome components are organized in a similar fashion. The 10 ␮g/ml A22 for 70 min, MreB coils disappeared although rod other components were identified by immunofluorescence micro- shape was retained (data not shown). Under these conditions, the scopy, using purified anti-RhlB antibody or antibody directed RNaseE coiled structures were preserved (Fig. 2 J). Thus, mainte- against an HA tag fused to enolase or PNPase. This showed that the nance of the cytoskeletal-like organization of RNaseE is indepen- three other degradosome proteins were also organized in extended dent of the helical MreB cytoskeleton. coiled structures (Fig. 2 D–F). The RhlB, enolase, and PNPase MinD also forms helical cytoskeletal structures along the length structures wound around the cell and extended from one cell pole of E. coli cells (3). The fact that the yeast two-hybrid study suggested to the other, thereby resembling the structure formed by RNaseE. an interaction between MinD and RNaseE suggested that the Thus, all of the known E. coli RNA degradosome components RNaseE coiled structures might be secondarily associated with the are organized into similar cytoskeletal-like helical structures within MinD cytoskeleton. We therefore determined the localization the cell. pattern of RNaseE-HA in a ⌬minCDE strain. As shown in Fig. 2K,

1668 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610491104 Taghbalout and Rothﬁeld Downloaded by guest on October 1, 2021 fusely localized throughout the cytoplasm (Fig. 3 AЈ–CЈ). The difference in localization pattern of RNaseE domains was not due to differences in cellular concentrations of the Yfp-labeled proteins as shown by quantitation of cellular fluorescence. We conclude that the RNaseE determinant responsible for membrane association and helical cellular organization lies between amino acids 418 and 602 of the RNaseE protein. This is consistent with previous ImmunoGold electron microscopy studies showing a peripheral localization of RNaseE in wild-type cells and in a mutant strain expressing RNaseE(1–602) (24). Interestingly, the localization determinant of RNaseE overlapped the RNaseE domain that interacts with MinD in yeast two-hybrid assays [RNaseE(378–659)] (Fig. 1).

Effects of Loss of RNaseE Helical Organization. To determine the functional significance of the cytoskeletal-like organization of 1–417 RNaseE, we compared the rne mutant strains AT8 [Prne-rne ] 1–659 and AT14 [Prne-rne ]. As described above, cells expressing RNaseE(1–417) are incapable of forming the coiled cellular structures (Fig. 2 H and I and 3BЈ) whereas cells that express RNaseE(1– Fig. 3. Cellular localization of Yfp-labeled RNaseE domains. Differential inter- 1–377 659) (Fig. 2G) show a helical pattern that resembles the structure ference contrast and YFP images are shown. (A) MC1000/Plac-rne ::yfp.(B) ⌬rne/P -rne1–417::yfp.(C) AT8/P -yfp::rne660–1061.(D) AT8/P -yfp::rne378–659.(E) formed by full-length RNaseE. lac lac lac 1–417 418–602 AT8/Plac-yfp::rne . (Scale bars: 2 ␮m.) AT8 [Prne-rne ] cells showed the following abnormalities, 1–659 which were not seen in AT14 [Prne-rne ] or in cells that expressed full-length RNaseE. First, AT8 cells grew slowly, with generation the RNaseE helical distribution pattern was maintained in the times in rich medium that were Ϸ55%, 43%, and 35% (at 30°C, absence of Min proteins. Thus, the cytoskeletal-like organization of 37°C, and 42°C, respectively) of the generation times of either strain

RNaseE is independent of the MinD cytoskeleton. The biological AT14 or the isogenic parent strain MC1000. Second, AT8 cells MICROBIOLOGY significance of the MinD–RNaseE interaction in the yeast two- showed a defect in cell division as shown by a mixed population hybrid system is currently under investigation. ranging from normal-length cells to long filaments (Fig. 4F). The division block was not mediated by the endogenous division inhib- RNaseE Domain Required for the Cytoskeletal-Like Organization of itor MinC or the SOS division inhibitor SulA (27–29) as shown by RNaseE. To define the RNaseE domain responsible for its the observation that filamentation was unaffected by the absence of cytoskeletal-like organization, different chromosomally en- either MinC or SulA (Fig. 4 E and G). Third, DAPI-stained AT8 coded RNaseE-HA fragments and plasmid-encoded Yfp- cells exhibited a chromosome segregation defect, manifested by labeled RNaseE fragments were examined (Fig. 1). (Throughout long nucleoid-free regions and extended stretches of DNA that Ј Ј the text, numbers in parentheses refer to the RNaseE domain presumably represent unseparated nucleoids (Fig. 4 E –G ). As extending between the indicated amino acid residues.) These expected from the abnormal nucleoid distribution pattern, strain 1–417 studies showed that the helical organization of RNaseE requires AT8 [Prne-rne ] produced many anucleate cells (arrows in Fig. 4 Ј Ј a domain located between amino acids 418 and 602 of the E –G ). The chromosome segregation defect was more pronounced RNaseE protein. To avoid the possibility that localization of at elevated temperatures. At 37°C, 7.7% of AT8 cells were anucle- plasmid-encoded Yfp-labeled RNaseE fragments may be af- ate and 28% (of 609 cells) had unseparated nucleoids. At 42°C, 7% fected by endogenous chromosomally encoded RNaseE, the of cells were anucleate and 88% (of 553 cells) had unseparated nucleoids. In contrast, AT14 [P -rne1–659], the wild-type parent fragments were expressed in the absence of full-length RNaseE. rne MC1000, and cells that filamented because of a defect in the Viability of ⌬rne cells that lack the full-length RNaseE protein unrelated FtsZ protein {WC1001 [ftsZ84ts]} showed normal chro- requires the RNaseE(1–417) domain that contains the endori- mosome segregation (Fig. 4 AЈ–DЈ and HЈ). Fourth, cells of strain bonuclease domain of the full-length protein (26). Therefore, the AT8 often contained large bulges that were frequently located at constructs were expressed either in ⌬rne cells or, when necessary 1–417 one or both cell poles and, in some cases, at the septal region (Fig. to retain viability, in strain AT8 [Prne-rne ], which expresses 4I). This was much more frequent at 42°C than at 37°C (34% vs. 1% chromosomally encoded RNaseE(1–417). of cells). The bulges usually contained chromosomal DNA, as Immunofluorescence studies of RNaseE(1–659)-HA (Fig. 2G) shown by DAPI staining (Fig. 4IЈ). This indicates that they are showed the same helical localization pattern as the full-length formed by outward bulging of the entire cell envelope and do not protein (Fig. 2C) whereas RNaseE(1–417)-HA was diffusely dis- result from an extrusion of the outer membrane due to a local defect tributed within the cytoplasm (Fig. 2 H and I). Equivalent results in attachment of inner membrane to the murein outer-membrane were obtained in fluorescence localization studies of RNaseE(1– layer (30, 31). We presume that a local weakening of murein, Ј 417)-Yfp (Fig. 3B ) and RNaseE(1–659)-Yfp (data not shown). probably at division sites, leads the three cell envelope layers to This suggested that the determinant of the helical distribution bulge outward because of the turgor pressure of the cell. pattern of RNaseE includes sequences located between residues The defects in growth, cell division, and chromosome segregation 1–417 417 and 659 of the protein. Evidence that RNaseE(418–602) was in AT8 [Prne-rne ], where the cytoskeletal organization is absent, 1–659 sufficient to form the membrane-associated coiled structure came were not observed in strain AT14 [Prne-rne ] in which the helical from a study of Yfp-labeled fragments that included all or part of RNaseE pattern was unperturbed (Fig. 4 CЈ and DЈ). Taken the RNaseE(418–659) domain. As shown in Fig. 3DЈ, Yfp-labeled together these results suggest that the cytoskeletal-like organization RNaseE(378–659) was peripherally localized and formed coiled of RNaseE may play a significant role in its cellular function, structures, although the structures were somewhat less clear than although the possibility that the defects are related to some other with RNaseE(1–659) (Fig. 2G). Yfp-RNaseE(418–602) showed a effect of deleting the RNaseE(418–602) cytoskeletal localization similar localization pattern (Fig. 3EЈ). In contrast, RNaseE(1–377)- domain has not been excluded. Yfp, RNaseE(1–417)-Yfp, and Yfp-RNaseE(660–1061) were dif- It is known that the cellular concentration of RNaseE is nega-

Taghbalout and Rothfield PNAS ͉ January 30, 2007 ͉ vol. 104 ͉ no. 5 ͉ 1669 Downloaded by guest on October 1, 2021 Fig. 4. Phenotypic abnormalities associated with the loss of RNaseE helical organization. Cell morphology is shown by differential interference contrast, and 1–659 DNA distribution is shown by DAPI fluorescence. (A, AЈ, B, and BЈ) MC1000 grown at 37°C (A and AЈ) or at 42°C (B and BЈ). (C, CЈ, D, and DЈ) AT14 [Prne-rne ] 1–417 1–417 grown at 37°C (C and CЈ) or at 42°C (D and DЈ). (E and EЈ) AT43 [⌬sfiA, Prne-rne ] grown at 37°C. (F, FЈ, I, and IЈ) AT8 [Prne-rne ] grown at 37°C (F and FЈ) 1–417 or at 42°C (I and IЈ). (G and GЈ) AT9 [⌬minB, Prne-rne ] grown at 37°C. (H and HЈ) WC1001 [ftsZ84ts] grown for3hat42°C. The arrows show positions of anucleate 1–417 cells; the arrowheads mark the nucleoid-free regions; a shows anucleate cell; b shows the bulges in AT8 [Prne-rne ] cells grown at 42°C. (Scale bar: 2 ␮m.) (J) 1–659 1–417 Immunoblot of total protein (54) of AT25 [Prne-rne::HA] (lane 1), AT27 [Prne-rne ::HA] (lane 2), AT28 [Prne-rne ::HA] (lane 3), and AT1/pRNE31 1–417 [⌬rne/Plac-rne ::HA] grown in the presence of 0 ␮M IPTG (lane 4), 1 ␮M IPTG (lane 5), 10 ␮M IPTG (lane 6), 100 ␮M IPTG (lane 7), or 1 mM IPTG (lane 8). Molecular masses are indicated in kDa. Thirty micrograms (lanes 1 and 2) or 5 ␮g (lanes 3–8) of protein was loaded.

tively regulated by its ability to degrade its own gene transcript (32). functions and in cell membrane and cell wall organization. For This autoregulation seems to be lost in the rne1–417 truncation example: MreB and MreB homologs appear to regulate the mutant, where the concentration of RNaseE(1–417) was 15- to organization of murein biosynthetic enzymes and thereby reg- 20-fold higher than RNaseE(1–659) or full-length RNaseE in cells ulate the shape of rod-shaped cells (34); MreB is also required in which the rne chromosomal copy was substituted by rne1–417::HA, for several aspects of differentiation of the cell poles (5, 6, 35); rne1–659::HA,orrne::HA (Fig. 4J, lanes 1–3). To exclude the the MinD cytoskeleton and its associated proteins are respon- possibility that the increased level of RNaseE(1–417) was responsible for placement of the bacterial division septum at mid-cell 1–417 sible for the abnormal phenotypes observed in AT8 [Prne-rne ], (3); the intermediate filament protein crescentin regulates cell we varied the cellular concentration of RNaseE(1–417) by express- curvature in Caulobacter crescentus (36); and the actin homolog ing rne1–417 under control of the lac promoter. This showed that the MamK is required for positioning of the membrane-bounded phenotypic defects were not corrected when the cellular concen- magnetosome organelles of Magnetospirillum magneticum (37). tration of RNaseE(1–417) was reduced to wild-type levels. Bacterial cytoskeletal elements also participate in movement of A decrease in the endoribonuclease specific activity of the nucleoids and plasmids within the cytoplasm, as shown by the RNaseE(1–417) mutant protein, suggested by studies of roles of MreB and ParM in chromosome and plasmid segrega- RNaseE(1–410) (33), could play a role in the phenotypic defects tion (7, 8, 38). In the present work, the finding that the of the rne1–417 strain. If a decrease in total cellular RNaseE components of the E. coli RNA degradosome show a charac- activity were responsible for the abnormal phenotype of AT8 teristic cytoskeletal organization indicates that cytoskeleton- 1–417 [Prne-rne ], an increase in cellular concentration of associated protein complexes also participate in reactions that RNaseE(1–417) would be expected to correct the defects. We modify cytoplasmic molecules, such as RNA processing and therefore increased the cellular RNaseE(1–417) concentration degradative reactions. This represents a previously unrecognized 1–417 (Fig. 4J) by expressing rne under Plac control. This failed to role for the cytoskeleton in the life of the cell. correct the abnormal phenotype over a 100-fold range of con- The mechanism responsible for the cytoskeletal organization centrations (data not shown), suggesting that the morphological of RNaseE is not known. Formation of the filamentous RNaseE defects are not due to a decrease in total cellular RNaseE structure within the cell could be an inherent property of the activity. Further proof for this will require identification and protein itself, based on an ability to self-assemble into filamen- study of a rne mutation that interferes with the helical cytoskel- tous structures in a manner similar to MreB and MinD (39, 40). etal organization of RNaseE without affecting its enzymatic Yeast two-hybrid studies have identified self-interacting do- activity. mains in RNaseE that could participate in such a self-assembly system (41). The domains include the RNaseE(418–602) region, Discussion which we show is required for the cytoskeletal-like organization The fact that bacteria contain several cytoskeletal elements that of RNaseE. However, until it is directly shown that RNaseE can impart long-range order to the cell suggests that these structures actually polymerize into extended filaments, the possibility of a play important roles in the life of the organism. In several cases, self-assembling RNaseE system remains conjectural. The finding the cytoskeletal elements participate in membrane-associated that the RNaseE structures were present in cells that lacked

1670 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610491104 Taghbalout and Rothﬁeld Downloaded by guest on October 1, 2021 MinD and in cells in which MreB cytoskeletal structures were (45, 46), and by the MinCDE cytoskeletal structure required for disrupted indicates that maintenance of the cytoskeletal-like division site localization (3). organization of RNaseE is independent of these cytoskeletal The present results suggest a role for the cytoskeleton in the systems. However, RNaseE might still interact with MinD or regulation of metabolic functions that take place within the MreB for other purposes despite the fact that the interactions are bacterial cytosol, in this case RNA processing and degradation. not needed for maintenance of the RNaseE helical structure. Because of its potential usefulness, it is likely that other cellular The significance of the MinD–RNaseE interaction in the yeast systems may use a similar strategy to provide the cell with two-hybrid system requires further study. It is also possible that additional means of regulating important cell functions. another, as yet unidentified, helical cytoskeletal system plays the role of a template or lattice for assembly of the RNaseE coiled Materials and Methods structures. Strains, Plasmids, Media, and Growth Condition. E. coli strains were ImmunoGold electron microscopy has shown that RNaseE is grown in LB medium (47) to which 100 ␮g/ml ampicillin, 25 localized at the cell periphery (24). This implies that the helical ␮g/ml kanamycin, 30 ␮g/ml chloramphenicol, or 0.4% (wt/vol) structures described here are associated directly or indirectly glucose was added when indicated. All plasmid constructions with the cytoplasmic membrane. The ImmunoGold peripheral were introduced into E. coli DH5␣ (47) and then transferred localization pattern required the first 602 residues of RNaseE to another E. coli or yeast strain. Yeast strains were grown in (24). This domain includes the RNaseE(418–602) determinant, supplemented SD medium (Clontech Yeast Two-Hybrid Man- which is required for the cytoskeletal organization of the protein ual) or in rich medium adenine-supplemented yeast extract/ (Figs. 2G and 3). A direct association of RNaseE with the peptone/dextrose (48). Plasmids and strains are listed in membrane via the RNaseE(418–602) domain could be required supporting information (SI) Table 1, and the details of their for its cytoskeletal-like organization within the cell. This would construction are available upon request. Strains AT8 [Prne- 1–417 1–659 resemble the requirement for the MinD membrane-binding rne ] and AT14 [Prne-rne ] were constructed by the ␭ domain in formation of the helical MinD-related cytoskeletal -red recombination method (49). HA-epitope tagging was structures (42–44). However, at present there is no evidence that done as previously described (50), and the associated antibiotic RNaseE(418–602) possesses membrane-binding activity. The cassettes were eliminated by use of the FLP-expressing plasmid existence of other peripheral cytoskeletal elements without pCP20 (51). P1-mediated transduction was used to move known membrane-targeting domains (reviewed in ref. 1) indi- mutations to different strains (52). Growth rate was deter- cates that the membrane association of coiled cytoskeletal mined by OD at 600 nm. MICROBIOLOGY structures does not always require direct interaction with the membrane. Therefore, the membrane association of the RNaseE Yeast Two-Hybrid E. coli Genomic Library Construction and Screening. An E. coli genomic library for use in the yeast two-hybrid system structures could be mediated by other cellular elements. was constructed as fusions to the yeast Gal4 activation domain Loss of the RNaseE(418–602) cytoskeletal determinant was (AD) of vector plasmid pAGADT7 (Clontech, Mountain View, associated with both loss of the helical RNaseE structure and CA). One milligram of chromosomal DNA from E. coli PB103 significant defects in growth rate, cell division, chromosome was partially digested at 37°C for 65 or 140 min in the presence segregation, and cell morphology, despite the presence of the of ATP and 0.6 or 0.3 units of CviJI (CHIMERx, Milwaukee, essential RNaseE endoribonuclease domain [RNaseE(1–417)]. WI), respectively. CviJI is a blunt cutter that cleaves between GC The defects were reversed by an RNaseE fragment that included of the RGCY recognition sequence (R, purine; Y, pyrimidine). both the RNaseE endoribonucleolytic domain and the domain In the presence of ATP the enzyme cuts between almost every required for the RNaseE helical distribution pattern. These GC pair (RGCN and YGCY) (53). Partially digested DNA was observations suggest that the cytoskeletal-like organization of purified and fractionated on a 5–20% sucrose gradient at 25,000 RNaseE may play an important role in regulation of normal cell rpm (SW41 Ti rotor) for 17 h at 20°C with 200 ␮g of DNA loaded functions by regulating the RNA processing functions of the on each gradient. Fractions containing fragments with an aver- degradosome. Previous studies have shown an increased half-life age size of 0.8 kb were selected from each gradient (Ϸ0.2- to of several mRNAs and a defect in the processing of rRNA and 2.5-kb size range) and were used in a 1:6 vector:insert molar ratio tRNA in cells that express only the RNaseE(1–417) fragment for ligation into 60 ng of SmaI-digested and calf intestine alkaline (17), which, as shown here, fails to assemble into the cellular phosphatase (CIP)-treated pAGDAT7 vector (Clontech). Liga- cytoskeletal-like structures. It is likely that the changes in mRNA tion products were concentrated by ethanol precipitation in the degradation and RNA processing are responsible for the various presence of 1 ␮g of glycogen and used to transform ELECTRO- phenotypic defects of cells that express the RNaseE(1–417) MAX DH10B competent cells (Invitrogen, Carlsbad, CA). domain in the absence of the domain required for RNaseE Approximately 19 ϫ 106 colonies were collected and resus- cytoskeletal organization. Further work is needed to prove that pended in 80 ml of SOC medium with ampicillin (47). One-half these defects all result directly from loss of the cytoskeletal of the cell suspension was used to inoculate 800 ml of LB- organization of the protein. ampicillin medium and grown for 75 min at 37°C. The cells were These observations lead us to suggest that the cytoskeletal then used for plasmid DNA extraction using eight Qiagen-tip 500 organization of the degradosome serves to compartmentalize es- columns (Qiagen, Valencia, CA) to prepare the genomic plasmid sential RNA processing and degradative activities within the cell, library. The other half was aliquoted and saved at Ϫ70°C in the thereby playing an important role in the regulation of their cellular presence of 20% glycerol. activities. One function of compartmentalization may be to seques- Plasmid pMDB1, containing minD fused to the Gal4 binding ter the degradosome machinery away from sites of RNA synthesis, domain, was prepared as previously described (23). To screen for thereby preventing premature or uncontrolled degradation of cer- DNA coding for MinD-interacting proteins, competent yeast tain essential RNA substrates. Cytoskeletal compartmentalization cells [made by the lithium acetate method (48)] from a 165-ml could also bring in close proximity other components that act culture of AH109/pMDB1 were transformed with 180 ␮gofthe cooperatively to target specific RNA substrates to the degradosome plasmid library. The yeast transformants were screened for machinery. The use of the cytoskeleton to spatially compartmen- interaction on supplemented SD medium without leucine, tryp- talize proteins and protein complexes is illustrated by the helical tophan, adenine, and histidine to select for cells that contained MreB cytoskeleton, which interacts with MreC and with compo- both the bait (BD-MinD) and the prey (AD library) plasmids nents of the cell wall biosynthetic machinery in several organisms (Clontech Yeast Two-Hybrid Manual). AD plasmids containing

Taghbalout and Rothﬁeld PNAS ͉ January 30, 2007 ͉ vol. 104 ͉ no. 5 ͉ 1671 Downloaded by guest on October 1, 2021 genomic fragments were separated from the binding domain against RhlB (kindly provided by M. Cashel, National Institutes (BD-MinD) plasmid by transferring the plasmid DNA isolated of Health, Bethesda, MD) was purified by absorption to purified from each yeast clone to E. coli DH5␣ and selecting for the His-RhlB bound to PVDF membrane, followed by elution with ampicillin-resistant AD library plasmid. To isolate plasmid from 0.2 M glycine (pH 2) and renaturation with 1.5 M Tris base (pH yeast, cells of 1.5 ml of culture grown overnight at 30°C in SD 8.8). Alexa Fluor 488- and Alexa Fluor 594-conjugated goat medium without leucine and tryptophan were resuspended in 60 anti-rabbit and Oregon green- and Alexa Fluor 488-conjugated ␮lof67mMKH2PO4 (pH 7.5) in the presence of 100 units of goat anti-mouse secondary antibodies were used (Molecular lyticase (Sigma, St. Louis, MO) and then incubated for1hat Probes, Carlsbad, CA). For comparison of cellular concentra- 37°C. Eleven microliters of 20% (wt/vol) SDS was added, and the tions of Yfp-labeled proteins, images were collected by using the lysate was vigorously vortexed for 1 min. Plasmids were extracted Openlabs image acquisition program (Improvision, Lexington, by using the Qiagen miniprep protocol except that only 183 ␮l MA), total fluorescence was measured for 15 individual cells to of P1 buffer was added. The isolated AD plasmids were first determine relative cellular concentrations by using the same screened by PCR to eliminate plasmids that contained MinD- or software, and the mean concentration in the labeled cells was MinC-encoding DNA fragments because it was previously shown calculated for each protein sample. The concentrations of dif- that MinD interacts with MinC and with itself in the yeast ferent labeled proteins were considered equivalent if they dif- two-hybrid assays (22, 23). fered by Ͻ20%. 1–417 To test whether the abnormal phenotype of AT8 [Prne-rne ] Microscopy. E. coli cells containing plasmids coding for Yfp- strain can be reversed by varying the cellular concentration of ⌬ 1–417 labeled proteins were grown in the presence of 10 ␮M isopropyl RNaseE(1–417), AT1/pRNE31 [ rne/Plac-rne ::HA] was ␤-D-thiogalactoside. Labeled cells were examined by fluores- grown overnight in LB medium supplemented with ampicillin cence microscopy as previously described (54); images were not and then diluted to OD600 0.05 and grown for5hat37°C in subjected to deconvolution. For DAPI staining, cells were fixed LB-ampicillin medium supplemented with 0.1% glucose and 0 ␮ ␮ ␮ ␮ ␮ ␮ ␮ in the presence of 0.2% glutaraldehyde and 2% formaldehyde M, 1 M, 10 M, 50 M, 100 M, 250 M, 500 M, or 1 mM for 20 min at room temperature, washed three times with saline, IPTG. The cells were then fixed and stained with DAPI as and then stained with 2 ␮g/ml DAPI for 10 min on ice and described above. washed with saline solution before microscopy. Immunofluores- cence methods (55) were described previously except that 100 We thank Mary Osborn and Asis Das for helpful discussions; M. Cashel, mM phosphate buffer at pH 6.8, 6.6, 7.4, and 7.4 was used during S. Cohen (Stanford University, School of Medicine, Stanford, CA), S. Uzzau (Centre National de la Recherche Scientifique, France), and M. fixation for enolase, PNPase, RhlB, and RNaseE, respectively. Wachi (Tokyo Institute of Technology, Tokyo, Japan) for proving Fixation was done in the presence of 0.02% glutaraldehyde and antibodies, strains, and reagents; and S. Dove for suggesting CviJI 2% formaldehyde. Monoclonal mouse anti-HA tag (Sigma) was enzyme for library construction. This work was supported by National used to detect HA-tagged proteins. Rabbit antiserum directed Institutes of Health Grant GM R37-06032.

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