identiﬁcation and characterization of a developmentally … · the esha gene was strongly...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.10.3004–3015.2001

May 2001, p. 3004–3015 Vol. 183, No. 10

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of a DevelopmentallyRegulated Protein, EshA, Required for Sporogenic

Hyphal Branches in Streptomyces griseusJANGYUL KWAK,1* LEE ANN MCCUE,2 KRISTEN TRCZIANKA,1 AND KATHLEEN E. KENDRICK1†

Department of Microbiology, Ohio State University, Columbus, Ohio 43210,1 and Wadsworth Center for Laboratoriesand Research, New York State Department of Health, Albany, New York 122012

Received 20 September 2000/Accepted 28 February 2001

To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors inStreptomyces, we examined total proteins of Streptomyces griseus by two-dimensional gel electrophoresis. Amongfive proteins that were present at high levels during sporulation but absent from vegetative cells, two of theproteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. Thededuced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins frommycobacteria and a cyanobacterium, Synechococcus, that are abundant during nutritional stress but whosefunctions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain,suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The eshA gene was stronglyexpressed from a single transcription start site only during sporulation, and accumulation of the eshAtranscript depended on a developmental gene, bldA. During submerged sporulation, a null mutant strain thatproduced no EshA could not extend sporogenic hyphae from new branch points but instead acceleratedseptation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA isrequired for the growth of sporogenic hyphae and localization of septation and spore maturation but not forspore viability.

Streptomyces is a gram-positive bacterium that undergoesmorphological differentiation. In a nutritionally favorable con-dition, Streptomyces spores germinate to give rise to vegetativehyphae, which are characterized by filamentous, multigenomiccells called mycelia. Sporulation begins by the growth of aerialhyphae (or sporogenic hyphae when the sporulation process isinduced in liquid culture; 34), which subsequently undergoseptation to form chains of unicellular spores. We previouslyidentified two types of sporogenic hyphae during sporulationof Streptomyces griseus (22): we suggest the term “distal” sporo-genic hyphae for those that grow from the tips of preexistingvegetative filaments and the term “proximal” sporogenic hy-phae for those that grow de novo within the vegetative hyphaeat the onset of sporulation. Since both vegetative growth andspore formation are inherently polar processes similar to thoseobserved in yeast and filamentous fungi (18), formation ofsporogenic hyphae in Streptomyces is likely to require proteinsthat direct the location of sporogenic hyphae, establishment ofpolarity, and emergence of the sporogenic hyphae.

To understand the molecular details of Streptomyces sporu-lation, researchers have identified developmental genes pri-marily by complementation of nonsporulating mutants. Mostof the sporulation-specific genes that have been characterizedappear to encode regulatory functions. Some, such as bldA,which encodes tRNA (38), are required for the production ofaerial hyphae (35, 43) and antibiotics (3, 34, 43). Genes that

specifically regulate spore formation include the whi genes, sonamed because mutations in these genes commonly preventformation of the spore compartments and the spore-associatedpigment that is acquired late in development (11). Includedamong these proteins are WhiG, a s subunit of RNA polymer-ase (12), and WhiH, a putative transcription regulator of theGntR family (52). A second sporulation-specific s factor isencoded by sigF. WhiG is required for the intermediate eventof sporulation septation, whereas SigF is required for subse-quent maturation of the spores (49).

Complete understanding of the role of developmentally con-trolled regulatory factors requires the characterization of thetarget genes they regulate. A few candidates for the targetshave recently emerged. The whiH promoter is recognized byWhiG, a sporulation-specific s factor (52). The whiE genecluster encoding grey spore pigment is controlled by severalwhi genes in Streptomyces coelicolor (29). Sporulating culturesundergo cycles of synthesis and degradation of glycogen, andsome of the enzymes of glycogen synthesis are active in aerialhyphae (7). The ftsZ gene, which is required for septation in S.coelicolor (41) and is differentially regulated during sporulationin S. griseus (J. Kwak, unpublished data), would therefore be alikely target of a factor that regulates development.

Here we describe results from attempts to identify additionalsporulation-specific genes encoding enzymes or structural pro-teins that might depend on the Whi proteins or other regula-tory factors for their expression during development. For thesestudies, we have exploited the ability of S. griseus to undergosporulation while submerged in liquid culture during nutri-tional downshift (33) or phosphate starvation (31). Underthese conditions sporulation is relatively rapid and synchro-

* Corresponding author. Present address: Institute for StructuralBiology and Drug Discovery, 800 E. Leigh St., Virginia BiotechnologyPark, Richmond, VA 23219. Phone: (804) 828-7573. Fax: (804) 827-3664. E-mail: [email protected].

† Deceased.

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nous; sporogenic hyphae are evident at 4 h and continue toelongate for the next 6 h, septation occurs at approximately10 h, and spores mature during the subsequent 10 to 12 h (34).Our objectives were to use two-dimensional gel electrophoresisto identify sporulation-specific proteins, to characterize thegenes encoding these proteins, and to establish the mecha-nisms of their developmental regulation. Here we report onone such protein, designated P3, that contains a cyclic nucle-otide-binding domain required for growth of sporogenic hy-phae at an early stage of streptomycete morphogenesis. On thebasis of its role in sporulation, we rename P3 as EshA (exten-sion of sporogenic hyphae).

MATERIALS AND METHODS

Bacterial strains and plasmids. S. griseus NRRL B-2682 was used as thewild-type strain. SKK2025, -2026, and -2027 were three independent isolatescontaining the eshA null allele. SKK1015 was used as the class IIIA mutant,SKK1008 was used as the class IIIB mutant, and SKK1003 was used as the classIIIC mutant, as described previously (34). Escherichia coli DH5a was used forroutine plasmid construction and preparation. E. coli ET12567 (dam, dcm, andhsd; 39) was used to demethylate plasmid DNA prior to its methylation in vitroand its introduction into S. griseus (J. Kwak, unpublished). We used E. coli Top10(Invitrogen, Carlsbad, Calif.) to express His-tagged EshA for making antibodies.

pKK842 was constructed by ligating a 2.4-kb SalI fragment of genomic DNAfrom S. griseus NRRL B-2682 to pGEM-4Z (Promega). The EcoRI-HindIIIfragment containing this 2.4-kb SalI fragment was ligated to pIJ2925 (27) togenerate pKK847. pKK856 was constructed by ligating the 2.4-kb fragment frompKK847 as a BglII fragment into the BamHI site of the low-copy vector pXE4(26). pKK1416 contained eshA on an approximately 8.0-kb BamHI-HindIII frag-ment in pXE4. pKK1417 contained the same 8.0-kb fragment in pKK1400 inwhich a 1.8-kb Tsr cassette (60) was inserted as a BamHI fragment into the BglIIsite of pIJ2920 (27).

To fuse EshA to a hexahistidine tag, we used the vector pTrcHis-A (Invitro-gen). Two oligonucleotides (oligonucleotide 164, 59-AGTCGGATCCGCTAGCATGACTGTTGACTCGACCTCGGA-39, corresponding to nucleotides [nt]329 to 352 of the 2.4-kb SalI fragment and containing adjacent BamHI and NheIsites, and oligonucleotide 165, 59-AGAACGTCCAGGGTGACCTCGTC-39,corresponding to nt 801 to 779) were used to amplify the N-terminal codingregion of eshA from pKK842, to introduce NheI and BamHI sites upstream ofand in frame with the eshA start codon. This PCR product was ligated toBamHI-SacI-digested pUC18 to generate pKK852. The 1.6-kb SacI-HindIIIfragment from pKK842, containing the C-terminal coding region of eshA, wasligated to pTrcHis-A that had been digested similarly, to yield pKK853. The0.4-kb NheI-SacI fragment from pKK852 was ligated to similarly digestedpKK853 to form pKK855, which contained the hexahistidine coding sequencefused to the N-terminal coding sequence of eshA.

To construct the null allele of eshA, a PCR-amplified fragment was generatedby using an M13 reverse sequencing primer (59-TCACACAGGAAACAGCTATGA-39) as the upstream primer and an internal primer containing a BclI site(59-CGGGAGGTGATCACCTGCATCTGCG-39, corresponding to nt 456 to432 of the 2.4-kb SalI fragment) as the downstream primer. The amplificationproduct was digested with EcoRI and BclI and ligated to pKK847 that had beensimilarly digested. This ligation generated pKK2011, which contained a deletedversion of eshA comprising the first 116 nt and the last 515 nt of the eshA codingsequence. This plasmid was digested with BclI and ligated to a 1.3-kb apramycinresistance cassette (obtained from P. Solenberg and R. Baltz in pCZA263) frompKK974. This ligation generated pKK2012. The Apr-disrupted eshA allele wastransferred as a BglII fragment from pKK2012 to pKK1400, which contains the1.8-kb thiostrepton resistance cassette (60) in pIJ2920 (27). This yieldedpKK2014, which was used as described below to construct the eshA null mutant.

Growth and induction of sporulation. E. coli cultures were grown in Luriabroth (2) supplemented with ampicillin (100 mg/ml) or apramycin (100 mg/ml) asneeded. Starter cultures of S. griseus were grown in SpM (31) for 2 to 5 days togenerate spore suspensions that were then used as inoculum for induction ofsporulation. Cultures of bald mutants were treated similarly except that the SpMculture was 36 to 48 h old at the time of subculture. SpM agar, supplemented asneeded with apramycin (20 mg/ml) or thiostrepton (5 mg/ml), was used formaintenance of streptomycete strains. SpMR (3) was used for protoplast trans-formation. Trypticase soy broth (BBL Microbiology Systems), 2XYT (53), orLuria broth was used for isolation of genomic and plasmid DNA from S. griseus (24).

Two different methods were used to obtain sporulating submerged cultures ofS. griseus. To induce sporulation by phosphate starvation or nutritional down-shift, 50 ml of glucose-ammonia minimal medium supplemented with 1% caseinhydrolysate (U. S. Biochemicals; salt-free) was inoculated with 0.05 to 0.5 ml ofan SpM starter culture and was incubated at 30°C on a shaker (250 rpm) until theabsorbance at 500 nm reached 3.0. Then the culture was harvested by centrifu-gation at 22°C, was washed once with prewarmed medium lacking casein hydro-lysate (nutritional downshift) and phosphate (phosphate starvation), and wastransferred to 50 ml of identical prewarmed medium in a 250-ml flask. The timeof transfer was considered 0 h (equivalent to vegetative growth). To obtainsporulation as a consequence of nutrient exhaustion, 0.5 ml of SpM starterculture was inoculated into 50 ml of SpM, and incubation at 30°C and 250 rpmwas continued. Typically the culture ceased exponential growth at an A500 of 9,and free spores and spore chains were first evident 6 h later.

Protein analysis. To prepare crude extracts, vegetative or sporulating cultureswere harvested by centrifugation at 14,000 3 g for 15 min at 4°C. The cells werewashed once with 1 M KCl and were suspended in 50 mM Tris-HCl, pH 7.5. Cellswere disrupted in a French press (14,000 lb/in2), and the lysate was centrifugedat 14,000 3 g for 5 min. The supernatant was used as the crude extract. Proteinconcentration was determined by the ultraviolet absorbance method of Ehres-mann et al. (19). Equal amounts of protein in sodium dodecyl sulfate (SDS)sample buffer were applied, and the gels were run and stained according tostandard methods (2, 36).

To examine total proteins by two-dimensional gel electrophoresis (48), weextracted proteins by using modifications of published methods (20, 21). Cellsharvested from a 50-ml culture were suspended in 2 ml of prechilled extractionbuffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2). After disruption in a Frenchpress (14,000 lb/in2), the broken cells were centrifuged at 12,000 3 g for 5 minand the supernatant was saved. Deoxyribonuclease I (Sigma; 100 mg/ml) andribonuclease A (Sigma; 50 mg/ml) were added to the supernatant, and themixture was incubated at 4°C for 20 min. The digested extract was combined withan equal volume of 20% (wt/vol) trichloroacetic acid in acetone and kept at220°C for 30 min. The proteins were collected by centrifugation as above, werewashed twice with cold acetone, and were dried in vacuo for 5 min. The residuewas dissolved in 1 ml of solubilization buffer containing 9.8 M urea, 4% NonidetP-40 (U. S. Biochemicals), 1% Ampholine (pH 3.5 to 10; Pharmacia), 1%Pharmalyte (pH 2.5 to 5; Pharmacia), and 2 mM dithiothreitol.

The isoelectric focusing gel for the first dimension was prepared according tothe procedure of O’Farrell (48) with minor modifications (20). The gel wasprerun in a GT2 tube gel unit (Hoefer Scientific Instruments, San Francisco,Calif.) at 400 V for 30 min and at 800 V for 30 min. Protein (25 ml) was loadedand was focused at 800 V for 12 h. The gel either was equilibrated for 15 min inrunning buffer (36) or was frozen at 220°C and equilibrated immediately beforeelectrophoresis in the second dimension. To determine the pI, a tube gel was cutin 5-mm slices, and each slice was macerated in 0.5 ml of water. The pH of eachsuspension was then measured with a pH meter. After electrophoresis, the geleither was fixed in 50% methanol and 10% acetic acid and stained with Coo-massie blue R-250 or was immediately prepared for electrophoretic transfer(Idea Scientific Co., Minneapolis, Minn.) onto polyvinylidene difluoride mem-brane (Bio-Rad). CAPS-NaOH (pH 11.0; 10 mM)–10% methanol was used as atransfer buffer. The membrane was stained with a 0.1% (wt/vol) solution ofPonceau S in 1% acetic acid. Protein spots were excised from multiple mem-branes and were sent to the Harvard Microchemistry Facility for amino acidanalysis and sequence determination.

Anti-EshA antibodies were made by purification of soluble EshA from the Histag system (Invitrogen) according to the manufacturer’s instructions. EshA wasthe only protein evident in the preparation by polyacrylamide gel electrophoresis(PAGE) after elution with 500 mM imidazole in 50 mM Tris-HCl, pH 7.5, and0.3 M NaCl. A homogenate of the pure protein in polyacrylamide was used toimmunize chickens (Cocalico Biologicals, Reamstown, Pa.). Rabbit anti-chickenantibodies conjugated to alkaline phosphatase were from Jackson Immunore-search and were detected by using the chromogenic method (Boehringer Mann-heim).

To isolate total proteins for immunoblot hybridization, equal biomasses ofcells (0.25 to 1.0 ml of liquid cultures) were harvested by centrifugation fromdifferent growth stages (during sporulation by phosphate starvation, nutritionaldownshift, and SpM). The cell pellets were resuspended in 0.5 ml of 50 mMTris-HCl (pH 7.5)–10 mM MgCl2, 2 mg of lysozyme was added, and the suspen-sions were incubated for 37°C for 10 min. The extracts were combined with 23SDS sample buffer (2, 36), and 30 ml of each extract was loaded onto the wellsfor SDS-PAGE. To isolate membrane proteins from cytoplasmic and ribosomalfractions, we used a modification of the procedure of Mikulik and Janda (44).One milliliter of the starter culture (4-day culture in SpM) was inoculated into 50

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ml of SpM. The culture was harvested at 6 h after the culture reached an opticaldensity at 500 mm of 9.0. This time point is when the culture enters stationaryphase and starts to form sporogenic hyphae. The cells were harvested by cen-trifugation at 14,000 3 g for 10 min. The cells were washed once and resus-pended in 1.5 ml of buffer A (20 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 0.5 mMphenylmethylsulfonyl fluoride). The cells were disrupted by a French press at14,000 lb/in2. The cell lysate was centrifuged at 14,000 3 g for 10 min at 4°C ina Beckman JA-20 rotor to remove the cell wall and other debris. The crude lysatewas centrifuged at 44,000 3 g for 30 min at 4°C to remove some larger membranefragments. The pellet (P44) was resuspended in 1.5 ml of buffer A. The super-natant (S44) was carefully removed and was centrifuged at 150,000 3 g for 14 h.After centrifugation, the supernatant (S150) was removed and the pellet (P150)was resuspended in 1.5 ml of buffer B (buffer A 1 1 M NH4Cl) at 4°C for 1 h andwas layered over 10 ml of a 50% sucrose cushion (10 mM Tris-HCl, pH 7.4, 500mM NH4Cl, 10 mM MgCl2, 6 mM 2-mercaptoethanol). The cushion was cen-trifuged at 150,000 3 g for 10 h at 4°C. Four fractions (upper cushion, brownlayer, lower cushion, and pellet) were isolated after the centrifugation. Thepellets were dissolved in 1.5 ml of buffer B. Thirty microliters of each proteinfraction was loaded onto a slab gel for SDS-PAGE and Western hybridization.

DNA manipulation and analysis. Standard (2, 24, 53) or previously published(35) methods were used for analysis and manipulation of DNA fragments forcolony hybridization, plasmid DNA minipreparations, and transformations. ForSouthern analysis we used the Genius II system after transfer of the DNA to apositively charged membrane (Boehringer Mannheim). We generated nesteddeletions by using exonuclease III digestion (23) of pKK842 to determine thenucleotide sequence of both strands of the 2.4-kb SalI fragment according to thedideoxynucleotide method (53). We used Deep Vent polymerase (New EnglandBiolabs) for PCR amplifications according to the manufacturer’s recommenda-tions.

The single-stranded DNA used in the S1 nuclease protection experiments wasproduced by using Deep Vent polymerase with a single oligonucleotide that wascomplementary to the coding sequence. The downstream primers oligonucleo-tide 166 (59-TGGACTGCCGGGGCACTTCCAG-39, corresponding to nt 380 to359 of the SalI fragment) and oligonucleotide 167 (59-TCCTGCATCTGCGGGGCGGACTT-39, corresponding to nt 444 to 422) were used to produce runoffpolymerization. The plasmid pKK842 digested with EcoRI was used as a tem-plate. The reaction conditions were similar to those recommended by the man-ufacturer for amplification of double-stranded DNA, except that 2 ml of dimethylsulfoxide and 0.5 mg of linearized template DNA were combined in reactionbuffer containing 3 mM MgCl2, and the reaction proceeded through 60 cycles.The single-stranded fragment was purified by using diatomaceous earth resin (9)after electrophoresis on a 1% agarose gel.

RNA studies. RNA was purified from vegetative and sporulating cultures of S.griseus NRRL B-2682, SKK1003, SKK1008, and SKK1015 by a standard method(24) with some modifications as follows. Ten microliters of vegetatively growingcells (A500, 3.0) was harvested by centrifugation at 4°C and then was suspendedin 10 ml of modified Kirby mixture (24). The cell suspension was passed througha French pressure cell (14,000 lb/in2), and the lysate was collected in 12-mlpolypropylene tubes containing 5 ml of Tris-buffered phenol-chloroform (1:1, pH8.0). After vigorous vortexing for 1 min, the mixture was centrifuged at 7,700 3g for 10 min. The upper phase was reextracted, vortexed, and centrifuged. Theaqueous phase was recovered, and the RNA was precipitated, was digested withDNase (RNase-free; Boehringer Mannheim) for 2 h at 37°C, and then wasextracted and precipitated as described above and dissolved in water.

To determine the transcription 59 end point, we used the S1 nuclease protec-tion assay (24) by combining 50 mg of RNA with 100,000 cpm of end-labeled,single-stranded DNA probe. The sequencing reactions were prepared with thesame primer that had been used to make the probe.

Gene disruption. pKK2014 was passed through E. coli ET12567 to removemethyl groups from adenine and cytosine residues and subsequently was meth-ylated with a mixture of commercially purchased methyltransferases, mAluI andmSssI, and endogenous methyltransferases from S. griseus NRRL B-2682 (40).After overnight incubation at 30°C, the reaction mixture was extracted once withphenol-chloroform and twice with chloroform and then was precipitated withethanol. The methylated DNA was dissolved in water and was introduced intoprotoplasts of S. griseus NRRL B-2682 suspended in P buffer (24; J. Kwak,unpublished). Apramycin-resistant transformants were selected and werescreened for thiostrepton sensitivity; this phenotype was indicative of a double-crossover event. Of 150 apramycin-resistant transformants, 3 were sensitive tothiostrepton, indicating that recombination had occurred on both sides of theapramycin cassette. Three such strains, SKK2025, -2026, and -2027, were iden-tified.

Microscopy. Phase contrast microscopy was performed with a Zeiss D-7082microscope equipped with a 35-mm camera. Since streptomycete filaments growin all directions, we improved the viewing quality by applying 1-ml samples to theslides and spreading them as thinly as possible with coverslips prior to viewing at31,000 total magnification. TMax 400 film was used for the photographs. Eitherthe prints or the negatives were scanned and were imported as TIFF files intoCorel PhotoPaint 8 for cropping and then into CorelDraw 8 for labeling andassembly of composite photographs. The appearance of each TIFF file wasadjusted with Corel PhotoPaint 8 to resemble the microscopic view as closely aspossible.

RESULTS

Identification of sporulation-specific proteins. To identifyproteins that accumulate to high levels under sporulation con-ditions (phosphate starvation and nutritional downshift) butnot during vegetative growth and that may therefore dependon developmental genes, we compared total proteins of sporu-lating cells with those of vegetative cells by two-dimensionalPAGE after staining with Coomassie brilliant blue. By com-paring the protein populations from both sporulation condi-tions, we sought to exclude proteins that might have beeninduced as a result of the phosphate regulatory network. Fivesporulation-specific proteins (named P1, P2, P3, P4, and P5)were present up to at least 12 h after induction (Fig. 1); afterthis time, the thick walls made the maturing spores refractoryto breakage at high pressure in a French press (34). The mo-lecular weights and isoelectric points measured on two-dimen-sional PAGE are shown in Table 1. These proteins becamevisible at different times during the first 12 h of sporulation(Table 1).

Since our hypothesis that proteins P1 through P5 weresporulation specific implied that developmental mutants mightlack some or all of these proteins, we looked for their productionin class III nonsporulating mutants of S. griseus (3). The classIII mutants, which include bldA mutants (34, 35) as well asthose with mutations in other uncharacterized genes, havecomplex phenotypes. The colonies appear bald on an agarmedium that ordinarily supports luxuriant sporulation. Wheninduced to sporulate in liquid culture by phosphate starvationor nutritional downshift, the class III mutants prematurelyundergo nucleoid segregation, septation, and spore maturationthroughout the preexisting vegetative filaments (34). Ectopicdevelopment of septation and spore formation in the mutantsis accompanied by premature fragmentation of the nascentspore chains. Thick spore walls form in these mutants by 6 h(class IIIA and C) to 8 h (bldA [class IIIB]) of sporulation, fully4 to 6 h earlier than in the wild-type strain (34). Two-dimen-sional gel electrophoresis revealed that extracts prepared fromall class III mutant strains during the first 6 h of sporulationlacked P3 (EshA) and P4, whereas P1 and P2 were present inthe extracts. P5 was produced at high levels by 4 h of phosphatestarvation in the bldA mutant (Table 2), which is 4 h earlierthan in the wild-type strain. We chose P3 (EshA) for furtherstudy.

The eshA gene encodes a protein related to stress-inducedproteins of other bacteria but with a likely cyclic nucleotide-binding domain. The N-terminal amino acid sequence of EshAwas identified as TVDSTSEARLEVPRQ. From this sequencea degenerate oligonucleotide (59-GARGCSMGBCTSGARGTSCC-39, corresponding to the seventh through thirteenthamino acid of the sequence) was synthesized and was used toidentify the eshA gene by Southern hybridization. A 2.4-kb SalI

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fragment of genomic DNA from wild-type S. griseus hybridizedto the oligonucleotide. Two streptomycete open readingframes (ORFs) were found in the DNA fragment by the Framecomputer program (67). The first ORF encoded a polypeptideof 470 amino acids with a molecular mass of 52 kDa and anisoelectric point of 5.3. The second one was a partial ORF. Thecomplete ORF (GenBank accession no. L76204) correspondedto the eshA structural gene because (i) the N-terminal se-quence of the mature protein was identical to that deduced

from the nucleotide sequence (less the fMet); (ii) the calcu-lated isoelectric point of the deduced protein (5.3) was close tothat of EshA (5.5) on the basis of its migration in the firstdimension of two-dimensional gel electrophoresis; and (iii) thecalculated and observed molecular masses were identical (52kDa). The translation initiation codon of eshA occurred 11 ntdownstream of a strong ribosome-binding site (AGGAG; 32).Two alternative, secondary structures could be formed fromthe sequence immediately downstream of the TAG stopcodon: one contained two adjacent, imperfect inverted repeats(nt 1743 to 1766 and nt 1772 to 1799; DG 5 231 kcal/mol) andthe other contained a single, imperfect inverted repeat (nt 1743to 1796; DG 5 235 kcal/mol) resembling a factor-independenttranscription terminator (15).

If EshA is an important protein in streptomycete sporula-tion, then we would expect it to be present in diverse species.We therefore examined S. coelicolor and its close relative Strep-tomyces lividans for the occurrence of eshA-related DNA se-quences by Southern hybridization using a 1.5-kb SphI frag-ment that contained the eshA coding sequence, 31 nt ofupstream DNA, and 93 nt of downstream DNA. Both speciesshowed identical patterns: a 4.3-kb BamHI fragment, a 7.5-kbPstI fragment, and a 2.4-kb SalI fragment hybridized at mod-erate stringency ($ 75% identity) to the probe (data not shown).Additionally, a BLAST search of the partial genome sequenceof S. coelicolor (www.sanger.ac.uk/Projects/S_coelicolor/blast_server.shtml) revealed the presence of an ORF that is 76%

FIG. 1. Identification of sporulation-specific proteins by two-di-mensional PAGE in the wild-type strain of S. griseus. Total proteinprofiles from vegetatively growing cells (A) and from cells after 12 h ofphosphate starvation (B) are shown. Arrowheads mark five sporula-tion-specific proteins (P1 through P5). The numbers indicate molecu-lar mass standards in kDa. The proteins were stained with Coomassiebrilliant blue R-250. The proteins are more horizontally spread inpanel B than in panel A due to longer running during isoelectricfocusing.

TABLE 1. Accumulation of developmentally regulated proteins inthe wild-type strain of S. griseus during the first 12 h of sporulationa

Protein Mol mass(kDa) pl

Protein accumulation at sporulation time(h):b

0 (veg) 2 4 6 8 10 12

P1 82 5.2 2 2 1 1 1 1 1P2 55 5.3 2 2 6 6 6 1 1P3 (EshA) 52 5.5 2 2 1 1 1 1 1P4 52 5.5 2 2 6 1 1 1 1P5 36 5.2 2 2 2 2 1 1 1

a Sporulation was induced by both phosphate starvation and nutritional down-shift in separate experiments.

b 1, protein present at a high level; 6, protein present at a low level; 2,protein absent; veg, equivalent to vegetative growth.

TABLE 2. Accumulation of developmentally regulated proteins inthe class III bald mutantsa

Protein

Protein accumulation at sporulation time (h):b

0 (veg), for strainsSKK1015, 21008,

and 21003

4, for the indicated strain

SKK1015 SKK1008 SKK1003

P1 2 1 1 1P2 2 6 6 6P3 (EshA) 2 2 2 2P4 2 2 2 2P5 2 2 1 2

a Sporulation was induced by both phosphate starvation and nutritional down-shift in separate experiments.

b 1, protein present at a high level; 6, protein present at a low level; 2,protein absent; veg, equivalent to vegetative growth.



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FIG. 2. Alignment of the amino acid sequence of S. griseus EshA (Sg EshA) with homologous proteins and the cNMP-binding domain. (A)Alignment with MMP-1 (SwissProt accession no. P46841), SrpI (SwissProt accession no. Q55032), and S. coelicolor EshA (Sc EshA; www.sanger.ac.uk/Projects/S_coelicolor/blast_server.shtml). (B) Alignment of the cNMP-binding domain. The cNMP-binding domain sequence given is themost probable amino acid at each position for the cNMP-binding superfamily described by McCue et al. (42). The shaded amino acids in rectanglesrepresent amino acid identities, and bold fonts show similar amino acids (A and G; M, I, L, and V; F, W, and Y; D and E; N and Q; K, R, andH; S and T) that are present in at least three of the four proteins. The seven amino acids shown to be involved in cNMP binding are marked withasterisks.



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identical (85% similar) to the deduced amino acid sequence ofS. griseus eshA (Fig. 2A).

A BLAST 2.0 search of the nonredundant database at theNational Center for BioTechnology Information (NCBI) (Fig.2A; 1) revealed that the deduced amino acid sequence of theeshA gene shared significant similarity with a mycobacterialprotein (MMP-1 from Mycobacterium leprae [SwissProt acces-sion no. P46841] and a 35-kDa protein from Mycobacteriumavium complex [SwissProt accession no. Q48899]) and withSrpI from the cyanobacterium Synechococcus PCC7942(SwissProt accession no. Q55032) (61). Although the functionof neither orthologous protein is known, MMP-1 is immuno-dominant in infected hosts (65) and SrpI is the deduced prod-uct of the third gene of a three-gene operon that is stronglyexpressed during sulfur starvation (47). Alignment of theseproteins showed that all three shared extensive similarity at theN terminus and within the C-terminal half (Fig. 2A). The Cterminus of each may contain a 14- to 17-amino-acid transmem-brane anchor (PHDhtm program; 51).

Noteworthy in this alignment was a domain in EshA that wasabsent from MMP-1 and SrpI (Fig. 2A). Multiple sequencealignment (42) and domain searches (4) revealed significanthomology of this domain with cyclic nucleotide (cNMP)-bind-ing domains from both procaryotic and eucaryotic cNMP-bind-ing proteins (Fig. 2B). This family of proteins contains those inwhich the activity is modulated by a cNMP, such as E. coli Crp,cNMP-dependent protein kinases, and gated ion channels (42,56). These cNMP-binding proteins have five glycine residuesbelieved to be important for formation of the cNMP-bindingpocket and two conserved residues that interact with thecNMP (Fig. 2B; 42). Dot plot analyses (The University ofWisconsin Genetics Computer Group Program; 16) alsoshowed strong similarity to the cNMP-binding domains of thecatabolite activator protein from Haemophilus influenzae andthe cGMP-dependent protein kinase from Drosophila melano-gaster (data not shown). Computer analyses using PROBE (46)and Classifier (50) also demonstrated that EshA shared ho-mology with procaryotic and eucaryotic proteins containingcNMP-binding domains.

EshA copurifies with the membrane. To confirm the expres-sion of EshA during sporulation, we performed Western hy-bridization experiments using a chicken antibody raised againstrecombinant EshA. We induced submerged sporulation by us-ing the complex medium SpM (31), as well as by phosphatestarvation and nutritional downshift. As expected, EshA wasdetected in cell extracts of all sporulated cultures (Fig. 3A).EshA was absent in extracts prepared from all class III non-sporulating mutants that had been sporulated by the sameinduction methods (data not shown).

MMP-1 copurified with membrane fractions (65), and theprogram PHDhtm (51) showed that EshA, MMP-1 from my-cobacteria, and SrpI from Synechococcus contained putativetransmembrane anchor domains at their C termini. To testwhether EshA is a membrane protein, we separated the mem-brane fraction from cytoplasmic and ribosomal fractions byusing differential centrifugation and detected EshA by usingWestern hybridization. EshA was much more abundant in theprotein extracts containing membrane fraction and large mem-brane fragments (Fig. 3B, lanes 1, 2, 3, 5, and 8) than in thecytoplasmic or ribosomal fractions (Fig. 3B, lanes 4, 6, 7, and 9).

eshA is developmentally regulated and depends on class IIIdevelopmental genes. Two eshA transcripts were identified bya high-resolution S1 nuclease protection assay using a single-stranded probe synthesized from oligonucleotide 167 (Fig. 4B).The 59 endpoint of the major transcript mapped 113 nt up-stream and the longer one mapped approximately 300 nt up-stream of the translation start codon (Fig. 4A and B). How-ever, the longer signal was not evident when the probesynthesized from oligonucleotide 166 was used, suggesting thatthe longer signal could be attributed to an artifact. The samesite was identified for the major transcript by using both probes(from oligonucleotides 166 and 167), and the endpoint wasidentical for cultures that were induced to sporulate by eitherphosphate starvation or nutritional downshift. The eshA tran-script was undetectable in RNA from vegetative hyphae butwas abundant in RNA extracted at 2 h of sporulation, theearliest time that was examined. A time course analysis showedthat the level of eshA transcript remained high through 12 h ofsporulation (Fig. 4B). The appearance of the eshA transcript by2 h of sporulation correlated well with the abundance of theprotein at 4 h. PCR amplification and nuclease protectionanalysis confirmed that the class III mutants contained theeshA gene but lacked the transcript (Fig. 4C). There was aputative promoter, including 210 (TAGTGT) and 235 (TTGGTC) regions measured from the 59 endpoint of the eshAtranscript, resembling the consensus sequence recognized byshrdB in Streptomyces (28, 57). The nucleotide sequence in theregions was identical in 16 of 20 positions to the sporulation-specific promoter (Pspo) of ftsZ in S. griseus (Fig. 5), which istranscribed at high levels only during sporulation (J. Kwak,unpublished).

FIG. 3. Detection of EshA by immunoblotting. (A) The accumu-lation of EshA during phosphate starvation (PS), nutritional downshift(ND), and SpM culture. The numbers indicate the hours of cultureafter shift into the sporulation induction medium. 0 shows the vegeta-tively growing cell immediately before the shift. V and S represent thevegetative cells and sporulating cells in liquid SpM culture (see Mate-rials and Methods). (B) Detection of EshA using immunoblotting fromcrude extract (lane 1), S44 (lane 2), P44 (large membrane fragments;lane 3), S150 (lane 4), P150 (lane 5), upper cushion (lane 6), brownlayer (lane 7), lower cushion (lane 8), and the pellet (lane 9; ribosomalfraction; 44).



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FIG. 4. Analysis of eshA transcripts by S1 nuclease protection. A single-stranded 400-nt DNA probe (including 20 nt of pGEM-4Z polylinkerand 328 nt upstream and 52 nt downstream of the translation start site of the eshA structural gene) was prepared by using oligonucleotide 166 fora primer extension in panel A. Oligonucleotide 167, which extended 116 nt downstream of the initiation codon, was used for the experiments shownin panels B and C. In each case the sequence ladder (A, G, C, T) was extended from the same primer and used as the size marker. (A) Identificationof the 59 end of the eshA transcript in the wild-type strain of S. griseus. P, RNA from cells that had been starved for phosphate for 4 h; N, RNAfrom cells that had been subjected to nutritional downshift for 4 h. The nucleotide sequence shown to the left of the autoradiogram is that of thesense strand. The asterisk marks the 59 end of the transcript, and the nucleotide numbers refer to positions in the 2.4-kb SalI fragment (GenBankaccession no. L76204). (B) Time course of eshA transcription during phosphate starvation. Total RNA was prepared from vegetative cells (V) andcells that had been starved for phosphate for 2, 4, 6, 8, 10, or 12 h. E. coli tRNA (E) served as the negative control. (C) Analysis of the eshAtranscript in the class III mutants during phosphate starvation. The sequence ladder and probe were prepared as described for panel B. Total RNAwas prepared from the wild-type strain of S. griseus and class III developmental mutants SKK1015 (class IIIA), SKK1008 (bldA; class IIIB), andSKK1003 (class IIIC) that were growing vegetatively (V) or starved for 2 or 4 h. The film was deliberately overexposed to demonstrate the absenceof signal in the mutants.



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An eshA mutant is defective in sporulation-specific growth.We constructed an eshA null allele by replacing the codingregion from Glu39 to Leu300 with an apramycin resistancecassette. The disrupted structure of the eshA gene was con-firmed by Southern hybridization analysis; all three isolatescontained the 2.9-kb SalI fragment expected for the disruptedgene and lacked the 2.4-kb SalI fragment characteristic of theintact eshA gene (data not shown). As expected, both frag-ments were present in a transformant resistant to both apra-mycin and thiostrepton. An immunoblot showed that the wild-type strain but not the null mutants produced EshA within 6 hafter the end of exponential growth in nutrient exhaustionmedium (data not shown).

We were not able to detect any difference in growth andsporulation between the null mutant and the wild-type strainwhen sporulation occurred on solid media. Like the wild-typestrain, the disrupted mutant produced streptomycin and grewat a normal rate (m 5 0.47 6 0.03 h21) in the complex medium.On buffered SpM agar, which permits abundant sporulation ofthe wild-type strain, the mutant sporulated to the same extentas the wild-type strain: the colony was as hairy as the wild-typestrain and the mutant produced abundant spores when ob-served with a microscope. Therefore, we turned to the sub-merged sporulation system (nutritional downshift) for moredetailed analysis. Under this condition, the wild-type strainfollows a reproducible time course of sporulation (22, 34). Inliquid culture, the sporulation yield (sonication resistanceunits) of the eshA mutant was the same as that of the wild-typestrain (5 3 109 to 7 3 109 sr/ml; 34). The spores from thesemutants did not have any defects in sonication resistance, ly-sozyme tolerance, or germination (31, 34) when compared tothe wild-type strain. The dramatic aspect of the mutant phe-notype during submerged sporulation was the aborted growthof branch sporogenic hyphae (Fig. 6). Phase contrast micros-copy showed that the defective branch sporogenic hyphae ini-tiated growth but failed to extend beyond a length of about 1to 2 mm, even after prolonged incubation. Instead, septationand spore maturation were accelerated at the tips of preexist-ing vegetative hyphae or de novo synthesized distal sporogenichyphae (Fig. 6). Frequently these stunted branch sporogenichyphae became swollen at their bases, perhaps indicative ofisotropic rather than polar growth. By 12 h, almost all of thedeformed branch sporogenic hyphae had detached from thevegetative hyphae (Fig. 6).

The eshA null mutant formed septate spore chains at thevegetative hyphal tips at 8 to 10 h. After 12 h of induction the

nascent spore chains of the mutant were fragmented and re-leased. In comparison, the wild-type strain requires 12 h toundergo septation and an additional 10 h to form fragmented,mature spore chains (Fig. 6; 34). The spore chains that formedin the null mutant were abnormal, however, because the distal“spore” in the chain generally appeared larger when comparedto other spores in the chain (Fig. 6). The septated region wasnoticeably longer than the “distal” sporogenic hyphae of thewild-type strain.

To demonstrate that the phenotype was caused by the de-letion of the eshA gene, we used two different strategies tocomplement the mutant. In the first case, we introduced thewild-type allele contained in either the 2.4-kb SalI fragment onpXE4 (pKK856) or the 8-kb BamHI-HindIII fragment con-taining eshA plus approximately 3.5 kb of upstream and 2.5 kbof downstream sequences (pKK1416). Then we comparedsporulation of these transformants with those that containedthe vector only. In the second approach, we constructedthe same 8-kb eshA fragment in the nonreplicative vectorpKK1400 at BamHI and EcoRI sites (pKK1417) and generateda single crossover strain by integrating into the chromosome byhomologous recombination at the eshA locus. Because thepresence of thiostrepton in the medium (to select for the main-tenance of the plasmid) lengthened the time required for de-velopment of both the wild-type strain and the mutant, weexcluded thiostrepton from the growth and nutritional down-shift media. At the end of the experiment, the viable count ofspores (measured as sonication-resistant units per ml; 34)showed that thiostrepton resistance was maintained in morethan 90% of the spores. This indicated that there was little ifany loss of the plasmid during the course of the experiment.The null mutant was complemented by the eshA gene in trans;branch sporogenic hyphae formed to the same extent as in thewild-type strain (data not shown). Since the complementingfragment lacked a complete downstream ORF, we concludethat the mutant phenotype was caused by the lack of EshA.

DISCUSSION

EshA is a stress-response protein. EshA is an abundantprotein that accumulates during sporulation induced by phos-phate starvation and nutritional downshift in S. griseus. Similarproteins are present in a cyanobacterium and two species ofmycobacteria. Although the functions of these homologousproteins are not known, they are produced abundantly whenthe cells experience nutritional stress: SrpI when Synechococ-

FIG. 5. The nucleotide sequence homology of the promoter regions between eshA and ftsZ. The shaded sequences mark the similar regions.The bold letters inside the shaded boxes show the putative 210 and 235 regions. n indicates the spacings between the putative 210 and 235regions. 11 indicates the 59 end of transcripts from both genes detected by high-resolution S1 nuclease mapping.



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cus encounters sulfur stress and MMP-1 during infection by M.leprae, which multiplies in phagosomes of the infected host(65). Since EshA is also made during nutritional stress, wehave proposed that EshA, SrpI, and MMP-1 define a newfamily of bacterial stress-response proteins (61). MMP-1 isbelieved to be a membrane protein and is routinely isolated asmultimers in excess of 20 subunits (65; W. J. Britton, personalcommunication). Our results also showed that EshA localizesto the cell membrane (Fig. 3B) and possibly exists as largemultimers (J. Kwak, unpublished). A putative transmembraneanchor in EshA, MMP-1, and SrpI was found by using the

program PHDhtm (51). In light of its abundance and andlocalization to the membrane, we speculate that EshA may bepart of a structural element. There is no EshA ortholog in E.coli, Bacillus subtilis, or other procaryotes whose genomes havebeen completely sequenced. A gene apparently homologous toEshA was also present in S. coelicolor and S. lividans, twospecies that are not closely related to S. griseus (32).

EshA is a determinant of growth of sporogenic hyphae. Thisstudy demonstrated that EshA is required for the maintenanceof growth of sporogenic hyphae from de novo branches and forproper formation of distal sporogenic hyphae, since the ab-

FIG. 6. Phase-contrast photomicroscopy of the eshA null mutant (SKK2025) and the wild-type strain of S. griseus after 6, 8, and 12 h ofsporulation induced by nutritional downshift. Sporogenic hyphae emerging from new branches (Br) and from preexisting vegetative hyphae (Tip)are marked. SC marks the spore chains. The bar represents 6.0 mm. No differences were apparent in vegetative cultures of the mutant and wild-typestrains.



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sence of EshA resulted in abortive growth of branch sporo-genic hyphae. We do not exclude the possibility that EshA mayfunction as an inhibitory factor of septum formation in thepreexisting vegetative hyphae until sporogenic hyphae are fullymature. However, EshA does not seem to be required forvegetative growth and branching, sporulation septum forma-tion, or spore maturation. Despite the absence of full-lengthsporogenic hyphae in the null mutants, spore chains and ma-ture spores formed from the preexisting vegetative hyphae.Consistently, the spores from these mutants did not have anydefects in sonication resistance, lysozyme tolerance, or germi-nation (31, 34) when compared to the wild-type strain. Thepremature septation and fragmentation demonstrated thatthese spore chains form ectopically by transformation of thevegetative filaments (Fig. 6 and 7). The ectopic septation andspore formation from the preexisting vegetative or distalsporogenic mycelia without mature growth of proximal sporo-genic hyphae (34) are also observed in the class III nonsporu-lating mutants that do not accumulate EshA during sporula-tion. Taking into consideration its possible existence as astructural component, and the phenotypes observed in theeshA null mutants and the class III mutants, it is conceivablethat this aspect of the null mutant phenotype could be causedby a high local concentration of factors required for septationand spore maturation that might mislocalize to the vegetativehyphae in the absence of sporogenic hyphal growth (Fig. 6 and7). For this reason, we named P3 as EshA (extension of sporo-genic hyphae). We speculate that the aberrant phenotype ofthe mutant observed in submerged culture also occurs on solidculture. The delicate morphological changes may not havebeen detected, since the cells harvested from solid cultureswere more heterogeneous and less synchronous.

Our assignment of EshA as a protein required for growth ofsporogenic hyphae in Streptomyces raises the question of theroles of the orthologous proteins in mycobacteria and Synecho-coccus. Limitation of Synechococcus for sulfur leads to theglobal activation of a number of genes whose products areresponsible for the utilization of alternative sources of sulfur(37), degradation of light harvesting centers (17, 55), and for-mation of a quiescent structure that requires extensive remod-eling of the cell membrane and wall (A. R. Grossman, personalcommunication). In view of the eshA null phenotype, we hy-pothesize that SrpI may be necessary for the morphologicalchanges that occur under this condition. Although mycobacte-ria are members of the Actinomycetales that grow with rudi-mentary branching, whether M. leprae displays morphologicalchanges during infection coincident with synthesis of MMP-1 isnot known (P. J. Brennan and W. J. Britton, personal commu-nication).

Developmentally regulated expression of EshA. Becausethe eshA transcript was evident only during sporulation, ex-pression of eshA is regulated at least in part at the transcrip-tional level. There was a single transcript 59 endpoint, regard-less of whether sporulation was induced by phosphatestarvation or nutritional downshift. This endpoint could alter-natively correspond to a site at which a longer transcript wasprocessed, but our results suggest that the 59 endpoint of theeshA transcript marks the transcription start site since therewas a putative promoter including 210 and 235 regions fromthe 59 endpoint and there was no evidence of a transcript 59end further upstream. Although either inverted repeat down-stream of eshA may act as a factor-independent transcriptionterminator, we do not yet know whether eshA and the down-stream ORF are cotranscribed.

FIG. 7. Diagram of alternative ways in which spores could be made in the null mutant. Initial formation of the sporogenic hyphae is unaltered(step 1). Subsequently, in the absence of EshA the proximal sporogenic hyphae form a bulbous structure characteristic of apolar growth.Simultaneously the apex of the distal sporogenic hypha becomes deformed (step 2). The spore structures arise either by ectopic septation andmaturation in the preexisting vegetative hyphae (step 3A) or by proper localization in the distal sporogenic hypha following a period of new growth(step 3B).



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The bald colony morphology of the class III mutants sug-gests that none of these mutations is allelic to eshA. Indeed, weknow that this is true for the class IIIB (bldA) mutants. More-over, the eshA null mutant produced streptomycin, unlike theclass III mutants. The absence of the eshA transcript in theclass III mutants was a consequence of the lack of gene ex-pression. This result shows that the class III mutants are de-fective in regulatory factors that mediate synthesis of EshA andother proteins necessary for sporulation and antibiotic produc-tion. One of these factors is a tRNA, the product of bldA. Therequirement of the class III developmental genes, includingbldA, for transcription of eshA prompted us to reexamine themorphology of a bldA mutant of S. griseus. We confirmed theprevious results (34) indicating that these mutants did notmake branch sporogenic hyphae, that the formation of sporechains in this strain occurred ectopically, and that this mutantunderwent premature fragmentation of developing sporechains. The observation indicates that the aberrant phenotypeof class III mutants is caused to a considerable extent by theabsence of EshA.

We speculate that the known sporulation-specific sigma fac-tors are not responsible for transcription of eshA. We canreadily rule out recognition by WhiG; whiG mutants areblocked at a later stage of sporulation because whiG mutantsform lengthy aerial hyphae but not spore compartments (11,30). Likewise, sF is not a candidate because it is required forthe relatively late events of spore maturation (49). However,the putative promoter for eshA including the 210 and 235regions is highly homologous to the consensus sequence rec-ognized by shrdB (28, 57), which is the essential sigma factor inStreptomyces (8). These results suggest that class III develop-mental genes, including bldA, regulate the transcription ofeshA during sporulation via a regulatory factor other than asigma factor.

The role of cyclic nucleotides in Streptomyces. EshA containsa cNMP-binding domain which is absent from the correspond-ing proteins from Synechococcus and Mycobacterium. Such adomain is present in proteins whose activity is modulated by acNMP but not in proteins that hydrolyze cNMPs (56). Theeucaryotic cGMP-dependent phosphodiesterases and cAMPreceptor proteins of Dictyostelium also contain distinctly dif-ferent cNMP-binding domains. EshA is a member of thecNMP-binding protein superfamily identified using PROBE(46) and Classifier (50). In E. coli Crp (62) and the regulatoryunit of bovine cAMP-dependent kinase (58), for which thethree-dimensional structure has been solved, seven invariantamino acid residues are required for interaction with thecNMP: the stability of an eight-stranded b-barrel likely de-pends on the five Gly residues; the ribose 29OH of cAMP ishydrogen bonded to Glu72; and Arg82 interacts with one ofthe exocyclic oxygens of the phosphate moiety (42). Theseseven residues are all conserved in EshA (42).

Cyclic nucleotides play widespread regulatory roles in eu-caryotes, where they govern processes such as polarized cellgrowth in filamentous fungi (6) and cation conductance inretinal photoreceptors (66). The known role of cyclic nucleo-tides in procaryotes is, up to now, much more limited. The onlyprocaryotic proteins known to bind cAMP are Crp and DnaA(25). In proteobacteria, which include enteric bacteria andmembers of phylogenetically closely related gram-negative

genera, Crp controls a variety of regulons, including thoseinvolved in catabolism (5), virulence (14, 64), and developmentof competence (10). With the exception of Crp, however, manyof the other members of the Crp-like protein family, such asFnr and FixK, have lost one or more of the amino acidsthought to play central roles in binding to cAMP (42) whileretaining their DNA-binding domains. Cyclic AMP has notbeen identified as an effector in the low-GC gram-positivebacteria. B. subtilis does not produce cAMP (P. Setlow, per-sonal communication) and lacks a gene resembling those en-coding either adenylate or guanylate cyclase. Cyclic diguanylicacid functions as a reversible allosteric activator of cellulosebiosynthesis in Acetobacter xylinum (63).

Streptomycetes accumulate cNMPs both intracellularly andextracellularly. Recent studies have shown that cAMP accu-mulates to a high extracellular level during the second expo-nential growth phase of S. griseus (45) and S. coelicolor (59).The gene encoding adenylate cyclase has been isolated from S.coelicolor (13). In addition to its inability to synthesize cAMP,a cya null mutant shows a complex phenotype that includesimpaired initiation of growth during spore germination andmorphogenesis; the latter defect is brought about by the in-ability to overcome the acidic environment generated by theextracellular accumulation of organic acids from glucose at theend of the first exponential growth period (59).

ACKNOWLEDGMENTS

We thank Keith Chater, Chuck Daniels, and Tina Henkin for crit-ically commenting on the manuscript. We also thank Tom Thompsonfor helpful discussion, R. Baltz and P. Solenberg for providing theapramycin-resistance cassette, D. MacNeil for providing the E. coliET12567 strain, Bill Lane of the Harvard Microbiochemistry Lab foramino acid sequence determination, Tim Vojt and Don Ordaz forphotoimaging processing, and Minho Kim for computer drawing.

This work was supported by grant MCB-9724038 from the NationalScience Foundation.

REFERENCES

1. Altschul, S. F., T. L. Madden, A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

2. Ausubel, F. M., A. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl. 1990. Current protocols in molecular biology. WileyInterscience, New York, N.Y.

3. Babcock, M. J., and K. E. Kendrick. 1988. Cloning of DNA involved insporulation of Streptomyces griseus. J. Bacteriol. 170:2802–2808.

4. Bateman, A., E. Birney, R. Durbin, S. R. Eddy, K. L. Howe, and E. LSonnhammer. 2000. The Pfam protein families database. Nucleic Acids Res.28:263–266.

5. Botsford, J. L., and J. G. Harman. 1992. Cyclic AMP in prokaryotes. Mi-crobiol. Rev. 56:100–122.

6. Bruno, K. S., R. Aramayo, P. F. Minke, R. L. Metzenberg, and M. Plamann.1996. Loss of growth polarity and mislocalization of septa in a Neurosporamutant altered in the regulatory subunit of cAMP-dependent protein kinase.EMBO J. 15:5772–5782.

7. Bruton, C. J., K. A. Plaskitt, and K. F. Chater. 1995. Tissue-specific glycogenbranching isoenzymes in a multicellular prokaryote, Streptomyces coelicolorA3(2). Mol. Microbiol. 18:89–99.

8. Buttner, M. J., K. F. Chater, and M. J. Bibb. 1990. Cloning, disruption, andtranscriptional analysis of three RNA polymerase sigma factor genes ofStreptomyces coelicolor A3(2). J. Bacteriol. 172:3367–3378.

9. Carter, M. J., and I. D. Milton. 1993. A simple method for DNA purificationon silica particles. Nucleic Acids Res. 21:1044.

10. Chandler, M. S. 1992. The gene encoding cAMP receptor protein is requiredfor competence development in Haemophilus influenzae. Proc. Natl. Acad.Sci. USA 89:1626–1630.

11. Chater, K. F. 1972. A morphological and genetic mapping study of whitecolony mutants of Streptomyces coelicolor. J. Gen. Microbiol. 72:9–28.

12. Chater, K. F., C. J. Bruton, K. A. Plaskitt, M. J. Buttner, C. Mendez, andJ. D. Helmann. 1989. The developmental fate of Streptomyces coelicolor



.org/D

ownloaded from

http://jb.asm.org/

hyphae depends upon a gene product homologous with the motility sigmafactor of Bacillus subtilis. Cell 59:133–143.

13. Danchin, A., J. Pidoux, E. Krin, C. J. Thompson, and A. Ullmann. 1993. Theadenylate cyclase catalytic domain of Streptomyces coelicolor is carboxy-terminal. FEMS Microbiol. Lett. 114:145–152.

14. de Crecy-Lagard, V., P. Glaser, P. Lejeune, O. Sismeiro, C. E. Barber, M. J.Daniels, and A. Danchin. 1990. A Xanthomonas campestris pv. campestrisprotein similar to catabolite activation factor is involved in regulation ofphytopathogenicity. J. Bacteriol. 172:5877–5883.

15. Deng, Z., T. Kieser, and D. A. Hopwood. 1987. Activity of a Streptomycestranscription terminator in Escherichia coli. Nucleic Acids Res. 15:2665–2675.

16. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set ofsequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395.

17. Dolganov, N., and A. R. Grossman. 1999. A polypeptide with similarity tophycocyanin alpha-subunit phycocyanobilin lyase involved in degradation ofphycobilisomes. J. Bacteriol. 181:610–617.

18. Drubin, D. G., and W. J. Nelson. 1996. Origins of cell polarity. Cell 84:335–344.

19. Ehresmann, B., P. Imbault, and J. H. Weil. 1973. Spectrophotometric de-termination of protein concentration in cell extracts containing tRNA’s andrRNA’s. Anal. Biochem. 54:454–463.

20. Garrels, J. I. 1983. Quantitative two-dimensional gel electrophoresis of pro-teins. Methods Enzymol. 100:411–423.

21. Granier, F. 1988. Extraction of plant proteins for two-dimensional electro-phoresis. Electrophoresis 9:712–718.

22. Hao, J., and K. E. Kendrick. 1998. Visualization of penicillin-binding pro-teins during sporulation of Streptomyces griseus. J. Bacteriol. 180:2125–2132.

23. Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates tar-geted breakpoints for DNA sequencing. Gene 28:351–359.

24. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M.Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward, and H. Schrempf. 1985.Genetic manipulation of Streptomyces. A laboratory manual. The John InnesFoundation, Norwich, Great Britain.

25. Hughes, P., A. Landoulsi, and M. Kohiyama. 1988. A novel role for cAMPin the control of the activity of the E. coli chromosome replication initiatorprotein, DnaA. Cell 55:343–350.

26. Ingram, C., M. Brawner, P. Youngman, and J. Westpheling. 1989. xylEfunctions as an efficient reporter gene in Streptomyces spp.: use for the studyof galP1, a catabolite-controlled promoter. J. Bacteriol. 171:6617–6624.

27. Janssen, G. R., and M. J. Bibb. 1993. Derivatives of pUC18 that have BglIIsites flanking a modified multiple cloning site and that retain the ability toidentify recombinant clones by visual screening of Escherichia coli colonies.Gene 124:133–134.

28. Kang, J. G., M. Y. Hahn, A. Ishihama, and J. H. Roe. 1997. Identification ofsigma factors for growth phase-related promoter selectivity of RNA poly-merases from Streptomyces coelicolor A3(2). Nucleic Acids Res. 25:2566–2573.

29. Kelemen, G. H., P. Brian, K. Flardh, L. Chamberlin, K. F. Chater, and M. J.Buttner. 1998. Developmental regulation of transcription of whiE, a locusspecifying the polyketide spore pigment in Streptomyces coelicolor A3(2). J.Bacteriol. 180:2515–2521.

30. Kelemen, G. H., G. L. Brown, J. Kormanec, L. Potuckova, K. F. Chater, andM. J. Buttner. 1996. The positions of the sigma-factor genes, whiG and sigF,in the hierarchy controlling the development of spore chains in the aerialhyphae of Streptomyces coelicolor A3(2). Mol. Microbiol. 21:593–603.

31. Kendrick, K. E., and J. C. Ensign. 1983. Sporulation of Streptomyces griseusin submerged culture. J. Bacteriol. 155:357–366.

32. Kim, E., H. Kim, K. H. Kang, Y. H. Kho, and Y.-H. Park. 1991. Completenucleotide sequence of a 16S ribosomal RNA gene from Streptomyces griseussubsp. griseus. Nucleic Acids Res. 19:1149.

33. Kroening, T. A., and K. E. Kendrick. 1987. In vivo regulation of histidineammonia-lyase activity from Streptomyces griseus. J. Bacteriol. 169:823–829.

34. Kwak, J., and K. E. Kendrick. 1996. Bald mutants of Streptomyces griseus thatprematurely undergo key events of sporulation. J. Bacteriol. 178:4643–4650.

35. Kwak, J., L. A. McCue, and K. E. Kendrick. 1996. Identification of bldAmutants of Streptomyces griseus. Gene 171:75–78.

36. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

37. Laudenbach, D. E., and A. R. Grossman. 1991. Characterization and mu-tagenesis of sulfur-regulated genes in a cyanobacterium: evidence for func-tion in sulfate transport. J. Bacteriol. 173:2739–2750.

38. Lawlor, E. J., H. A. Baylis, and K. F. Chater. 1987. Pleiotropic morphologicaland antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2). Genes Dev. 1:1305–1310.

39. MacNeil, D. J., K. M. Gewain, C. L. Ruby, G. Dezeny, P. H. Gibbons, and T.MacNeil. 1992. Analysis of Streptomyces avermitilis genes required for aver-mectin biosynthesis utilizing a novel integration vector. Gene 111:61–68.

40. Matsushima, P., and R. H. Baltz. 1994. Transformation of Saccharopolysporaspinosa protoplasts with plasmid DNA modified in vitro to avoid host restric-tion. Microbiology 140:139–143.

41. McCormick, J. R., E. P. Su, A. Driks, and R. Losick. 1994. Growth andviability of Streptomyces coelicolor mutant for the cell division gene ftsZ. Mol.Microbiol. 14:243–254.

42. McCue, L. A., K. A. McDonough, and C. E. Lawrence. 2000. Functionalclassification of cNMP-binding proteins and nucleotide cyclases with impli-cations for novel regulatory pathways in Mycobacterium tuberculosis. Ge-nome Res. 10:204–219.

43. Merrick, M. J. 1976. A morphological and genetic mapping study of baldcolony mutants of Streptomyces coelicolor. J. Gen. Microbiol. 96:299–315.

44. Mikulik, K., and I. Janda. 1997. Protein kinase associated with ribosomesphosphorylates ribosomal proteins of Streptomyces collinus. Biochem. Bio-phys. Res. Commun. 238:370–376.

45. Neumann, T., W. Piepersberg, and J. Distler. 1996. Decision phase regula-tion of streptomycin production in Streptomyces griseus. Microbiology 142:1953–1963.

46. Neuwald, A. F., J. S. Liu, D. J. Lipman, and C. E. Lawrence. 1997. Extractingprotein alignment models from the sequence database. Nucleic Acids Res.25:1665–1677.

47. Nicholson, M. L., M. Gaasenbeek, and D. E. Laudenbach. 1995. Two en-zymes together capable of cysteine biosynthesis are encoded on a cyanobac-terial plasmid. Mol. Gen. Genet. 247:623–632.

48. O’Farrell, P. H. 1975. High resolution two-dimensional electrophoresis ofproteins. J. Biol. Chem. 250:4007–4021.

49. Potuckova, L., G. H. Kelemen, K. C. Findlay, M. A. Lonetto, M. J. Buttner,and J. Kormanec. 1995. A new RNA polymerase sigma factor, sF, is requiredfor the late stages of morphological differentiation in Streptomyces spp. Mol.Microbiol. 17:37–48.

50. Qu, K., L. A. McCue, and C. E. Lawrence. 1998. Bayesian protein familyclassifier. Proc. Int. Conf. Intell. Syst. Mol. Biol. 6:131–139.

51. Rost, B., P. Fariselli, and R. Casadio. 1996. Topology prediction for helicaltransmembrane proteins at 86% accuracy. Protein Sci. 5:1704–1718.

52. Ryding, N. J., G. H. Kelemen, C. A. Whatling, K. Flardh, M. J. Buttner, andK. F. Chater. 1998. A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2). Mol.Microbiol. 29:343–357.

53. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

54. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

55. Schwarz, R., and A. R. Grossman. 1998. A response regulator of cyanobac-teria integrates diverse environmental signals and is critical for survivalunder extreme conditions. Proc. Natl. Acad. Sci. USA 95:11008–11013.

56. Shabb, J. B., and J. D. Corbin. 1992. Cyclic nucleotide-binding domains inproteins having diverse functions. J. Biol. Chem. 267:5723–5726.

57. Strohl, W. R. 1992. Compilation and analysis of DNA sequences associatedwith apparent streptomycete promoters. Nucleic Acids Res. 20:961–974.

58. Su, Y., W. R. Dostmann, F. W. Herberg, K. Durick, N. H. Xuong, L. TenEyck, S. S. Taylor, and K. I. Varughese. 1995. Regulatory subunit of proteinkinase A: structure of deletion mutant with cAMP binding domains. Science269:807–813.

59. Susstrunk, U., J. Pidoux, S. Taubert, A. Ullmann, and C. J. Thompson. 1998.Pleiotropic effects of cAMP on germination, antibiotic biosynthesis andmorphological development in Streptomyces coelicolor. Mol. Microbiol. 30:33–46.

60. Thompson, C. J., T. Kieser, J. M. Ward, and D. A. Hopwood. 1982. Physicalanalysis of antibiotic-resistance genes from Streptomyces and their use invector construction. Gene 20:51–62.

61. Triccas, J. A., N. Winter, P. W. Roche, A. Gilpin, K. E. Kendrick, and W. J.Britton. 1998. Molecular and immunological analyses of the Mycobacteriumavium homolog of the immunodominant Mycobacterium leprae 35-kilodaltonprotein. Infect. Immun. 66:2684–2690.

62. Weber, I. T., and T. A. Steitz. 1987. Structure of a complex of catabolite geneactivator protein and cyclic AMP refined at 2.5 A resolution. J. Mol. Biol.198:311–326.

63. Weinhouse, H., S. Sapir, D. Amikam, Y. Shilo, G. Volman, P. Ohana, and M.Benziman. 1997. c-di-GMP-binding protein, a new factor regulating cellulosesynthesis in Acetobacter xylinum. FEBS Lett. 416:207–211.

64. West, S. E. H., A. K. Sample, and L. J. Runyen-Janecky. 1994. The vfr geneproduct, required for Pseudomonas aeruginosa exotoxin A and proteaseproduction, belongs to the cyclic AMP receptor protein family. J. Bacteriol.176:7532–7542.

65. Winter, N., J. A. Triccas, B. Rivoire, M. C. Pessolani, K. Eiglmeier, E. M.Lin, S. W. Hunter, P. J. Brennan, and W. J. Britton. 1995. Characterizationof the gene encoding the immunodominant 35 kDa protein of Mycobacte-rium leprae. Mol. Microbiol. 16:865–876.

66. Wolbring, G., and P. P. Schnetkamp. 1996. Modulation of the calciumsensitivity of bovine retinal rod outer segment guanylyl cyclase by sodiumions and protein kinase A. Biochemistry 35:11013–11018.

67. Wright, F., and M. J. Bibb. 1992. Codon usage in the G1C2rich Strepto-myces genome. Gene 113:55–65.



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