The Streptomyces aureofaciens homologue of the whiG gene encoding a putative sigma factor essential for sporulation

Gene. 143 (1994) loll103 0 1994 Elsevier Science B.V. All rights

reserved.

101

037%1119/94/$07.00

GENE 07851

The Streptomyces aureofaciens homologue of the whiG gene encoding a putative sigma factor essential for sporulation (Recombinant

DNA; RNA polymerase;

sequence

Streptomyces coelicolor; Streptoverticillium griseocarneum;

comparison;

development)

Jgn Kormanec, Institute

Laura PottiEkovB and Bronislava

aeiuchov&

qf Molecular Biology, Slovak Academy of Sciences, 842 51 Bratisha,

Received by K.F. Chater:

14 October

1993; Revised/Accepted:

24 November/30

Slouuk Republic November

1993; Received at publishers:

27 January

1994

SUMMARY

A putative o factor-encoding gene, rpoZ, was cloned from Streptomyces aureofaciens. Its deduced protein product showed high similarity to the putative o factor encoded by the Streptomyces coelicolor whiG gene. Disruption of rpoZ blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores.

INTRODUCTION

The process of differentiation in species of the mycelial prokaryotic genus Streptomyces is controlled at several levels, among which the heterogeneity of 0 factors seems to play an important role (Chater, 1989). In St. coelicolor A3(2), the whiG gene, essential for sporulation, appears to encode an RNA polymerase o factor (Chater et al., 1989). Here we describe the identification, sequence analysis, and disruption of a putative St. aureofaciens o factor gene, rpoZ, closely similar to whiG.

EXPERIMENTAL

AND DISCUSSION

(a) Cloning and sequencing A St. aureofuciens CCM3239 (ATCC10762) genomic library (2-4-kb TaqI partially digested chromosomal fragments cloned into the ClaI site of the Escherichia coli plasmid pBR322) was hybridized with a degenerate Correspondence to: Dr. J. Kormanec, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovak Republic, Tel. (42-7)378-2432; Fax (42-7)372-316; e-mail: [email protected] Abbreviations: SSDI

aa, amino

0378-1119(94)00062-W

acid(s); B., Bacillus; bp, base pair(s);

dCTP,

reverse oligo probe S-GCSTYGATSAGSCC (where S = C or G and Y =C or T) encoding a peptide motif GLI(K,D,N,E)A, homologous to a part of the most conserved domain 2.2 of almost all (3 factors (Lonetto et al., 1992). Analysis of positive clones revealed four representatives, which were sequenced. In one case (Fig. l), sequencing revealed an ORF (rpoz), whose deduced product (278 aa, M, 30 714) showed extensive similarity to all conserved domains in o factors (Lonetto et al., 1992). The highest similarity was to the whiG-encoded putative o factors of St. coelicolor (Chater et al., 1989) and Sv. griseocarneum (Soliveri et al., 1993) (91 and 90.6%, respectively), and to flagellar and motility o factors (Helmann et al., 1988; Ohnishi et al., 1990; Starnbach and Lory, 1992). Alignment of these (5 factors (Fig. 2) shows extensive similarity mostly in conserved regions 2.4 and 4.2, proposed to be involved in the recognition of the - 10 and - 35 regions of promoters (Lonetto et al., 1992). This indicates that they all may recognize similar promoters. Indeed, two St. coelicolor promoters, dependeoxyCTP; E., Escherichia; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; P, Pseudomonas; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; R, resistance/resistant; RBS, ribosome-binding site(s); S., Salmonella; SDS, sodium dodecyl sulfate; St., Streprom~ces; Sr., Streptocerticilium; Th, thiostrepton; wt. wild type.

102 -

60

GGCCTCTCCGCCGGAGTGCACGAGCTGCTGCGAGGCACCGACGCC GLSAGVHELLRGEAQLVTDA

120

GCCGAGGTGG~~~GGTCGGCGACATCGGCGAGCTGGCACCCGAGCGCC~GGAC~G AEVVELVGDIGELAPERRGP

180

GTGCTCGCCCGCGA~CTCCTCCCCCGGACACCGTGCGCG~CTCGACGCGCTCCCCGCC VLARDLLPPDTVRVLDALPA

240

GGCCGGGCCGCGCGACTGGAGGAGATCACCCTCGCCGCGGCCACCGGTACCGATG~GTC GRAARLEEITLAAATGTDEV

300

ATCGGCAGACTGTACGMCTTCACTCTCTGGGG~CGTCG~C~CAG~CGA~GGCTGG IGRLYELHSLGFVERQGDGW

360

CGGTTGACCCCGCGCACGCCACGAGAGOOGGGCAATCC RLTPRTPREGAQSRATRRGG

120

CATTGACCTGGGGCGTTCCGGTGAAAGAGTGATG H l

.*o

GGCAGCCGCCGGGACCGAGCGCCAGGCGCCGGTCCCGGGCCGCCCGTGCGAGTCCGGGCG

540

Z.l-

MPQHTSGSDRAAVPPAAR--GSVRSTAPSSLEVL”RSYKDSGDF.RLREQL~LRYSPLVKY MPQHTSGSDRAAAPPAAR--GAARPPAPSTLDELWRSYKASGDERLRRQLILRYSPLVKY HPQ”TSGSDRAAIPPAARDGGSVRPPAPSTLDELWRSYK’lTGDERLREQLILRYSPLVKY MNSLYTAEGVMDKHS-LWQRYVPLVRR ~~SG~S~QAQNSQSQLIQRYAPL~R WQSLNYSD~~R~EWXDP~GDDL~~PL~ t ttt * . 2.4 2.2 2.3 VAGRVSVGLPPNVEQADFVSSGVFGLIDAI[EKFDIERSIKFE~AI~IRG~IDEL~ VAGRVSVGLPPNVEQADPVSSGIFGLIDAIEI(FDPARAIKFETYAI~I~G~IDEL~ VAGRVSVGLPPNVEQADFVSSGVFGLIDAIEKFDVDREIKFETYAlTRIRCAMIDELRAL EALRLQVRLPASVELDDLLQAlACGIOLLNAVDRYDALQGTLDELRSR AAKKYDAGKGASFETYAGIRIRGXLDEVRXG IAYRI.LGRLPASVQVFaDLnQACHICLLE “VGRIS“GLPKSV”KDDLHSLGXLGLYM”PLKNLTQPDLKFDTYASFR~RGAII~~RKE l *et **t**..t..t . ** t l . . tt *

-

-3 DWIPRSVRQ~VE~YAT~QLRRTPTEGEVAG~GIGV~~~SQLS~~ DWIPRS~Q~VE~YATL~~RRT~~EV~~GIALE~HSVFSQLS~AL DWIPRSVRQ~~~YATL~RLRRTPSESEVAV~GIAV~L~VFSQLSL~AL DWPRSVRRNAREVAQAMGQLEQELGRNATETEVARRLGIOVARSQLFSY DWAPRSVXRNTRMVTDAIRAIEARTGRDAKDHEVAAELQLSLEDYYGILSDTQGSRLYSF DWLPRTSREKTKKVFAAIEEQRYLRNVSPAEIAEELGMTVQDVVST”NEGFF~~SI *t.**. . ” * .t * *.* . . .

+++

r’ + CCGCTGCGCCCCCGCGCCATCCCGTACTCTTCGCGCGCACCGCGACACTTCCGTCACGCTAC . . .

600

++ ++-I GCTCCCAAoG~TCCCGCTCCGGCAAAGCOAAGCATGCC~~AGCACACCTCAGGGT~CGA . . . . HPQHTSGSD

660

CCGCGCTGCGGTGCCCCCCGCCCGCGGCAGCGGCCCT RAAVPPAARGSVRSTAPSSL

“I20

GGAGGTGCTCTGGCGCTCATACAAGGACTCGGACTCGGGCGACG~CGGCTGCGGGAGCAGCTGAT EVLWRSYKDSGDERLREQLI

780

CCTGCACTACTCGCCGCTGGTGAAGTACGTGGCGGGCCGGGTCAGCGTCGGCCTGCCGCC LHYSPLVKYVAGRYSVGLPP

**0

CAACGTGGAACAGGCCGACTTCGTCTCCTCCOGGGGTC~C~CC~ATCGACGCGATCGA NVEQADFVSSGVFGLIDAIE

PGO

GAAGTTCGACATCGRACGGTCCATCAAOTTCCAGACGAGACGTACGCCATCACCCGGATCCGCGG KFDIERSIKFETYAITRIRG

960

CGCGATGATCGACGAACTGCGGGCGCTCGACTGGATCCCGCGCTCGGTACGGCAG~GGC AHIDELRALDWIPRSVRQKA

,020

GCGCGCCGTGGAGCGGGCCTACGCCACCCTGGAGGC~CAG~GCGGCGGACCCCGACGGA RAVERAYATLEAQLRRTPTE

1080

11.0

GTTGTCCCTCGCCAACGTGGTCGCCCTCGAAGAGCTGCTGCACGTCGGCGGGGAGGGCGG LSLANVVALEELLHVGGEGG

1200

CGACCGCCTCTCCCTGATGGACACCCTGGAGGACCACGCCGCCGACGACCCCGTCGAGGT SLHDTLEDHAADDPVEV D R L

1260

GGCCGAGGACCGGGAGTTGCGCCGGCTOCTCGCACGGOC~G AEDRELRRLLARAINTLPER

1320

GAGGGCCAAGCTGGCCGACGTCGGCCGGTGAGCCGGTACC RAKLADVGRI

.

.

t

.

* t *.t

l . t.*

278 27s 280 239 247 254

2. Alignment of the aa sequences of Sr. uureofuciens &pox (Sa), St*. griseocurnrurtl (Sg) and St. coelicolor (SC) c?“~ (Soliveri et al., 1993; Chater

et al., 1989), S. typhimurittm (St) and

P. aeruginosa (Pa) oF

(Ohnishi et al., 1990; Starnbach and Lory, 1992), and B. suhtilis (Bs) c? (Helmann et al.. 1988). The asterisks indicate aa that are identical in all sequences: the dots indicate aa that are similar in all sequences (similar aa were: I,V,L,M; F,Y; N,Q: S,T; D,E; R,K). The subregions conserved among (J factors (Lonetto et al., 1992) are denoted by arrows above the sequence.

1380

11.0

CGTGCTGGGCGTGACCGAGAGCCGCGT~GC~AGATCCAT~~AG~T VLGVTESRVSQIHTKSVLQL

..

1480

Fig. 1. The nt sequence of the St. ~~r~~~~~~~~~rpoZ gene and flanking DNA. The deduced rpoZ gene product and the upstream S-truncated ORF are given in the singie-letter aa code in the second position of each codon. A presumptive RBS is indicated by solid dots and stop codons are marked by asterisks. The stretch of DNA that hybridized with the oligo probe is indicated by a horizontal black bar. The bent arrows encompass the rpoZ-upstream region of high similarity with Sf. c~elieo~[~r and St-. g~iseoc~rF~eu~ ~,~iG-upstream sequence. Crosses mismatches in St. co&color and small circles in Sn. sequences. This sequence has been deposited in GenBank/EMBL/DDBJ data bases under accession No. 90414. Methods: The nt sequence was determined in both strands after 3’-end labelling with PolIk and [u-~‘P]~CTP according to Maxam and Gilbert ( 1980). Compressions caused by a high percentage of G-C pairs were removed by running equilibrated gels at 75-C. Colony blot hybridization with an 14mer oligo mixed probe was performed at 30°C in

represent

griseocnrncurn

16% (v/v) formamide according to Ausubel et al. (1987). Two replica Hybond N filters (Amersham) were washed after hybridization in 2 x SSC (0.3 M NaC1/0.03 M Na,citrate pH 7.0)/0.1X SDS at 35°C and 40X’.

dent (directly or indirectly) on whiG, have sequence features resembling those of gF- or oD-dependent motilityrelated promoters (Tan and Chater, 1993). The striking similarity is in the N-terminal part (aa 52-68 in rpoZ) that is not usually conserved between different D factors. Moreover, all aligned proteins contain three conserved aa in this domain (aa GLP in position 66 of rpp0.Z) that are absent from all other identified o factors (Lonetto et al., 1992). Sequencing upstream from rpoZ revealed DNA encoding the C terminus of a protein with 50% identity to the product of a similarly located gene upstream from w~iG in St. coelicolor. The 209 bp of non-coding DNA between the upstream ORF and rpoZ contains sequence highly conserved with the equivalent regions of the St. coelicolor and Sv. griseocarneum whiG genes, flanked by nonconserved regions (Fig. 1). The functional significance of these sequences remains to be determined. (b) Disruption of the rpoZ gene The chromosomal copy of rpoZ was inactivated by a double cross-over employing the recently described method (Kormanec et al., 1993). A plasmid, with the tsr gene inserted in coding region of rpoZ, was used to trans-

103 form

St. aureofuciens protoplasts.

formants,

the

cross-over,

integration

resulting

In four

occurred

through

in the replacement

gene by the disrupted allele (confirmed hybridization). All four rpoZ-disrupted strains

had a white aerial mycelium,

grey of the parental

strain.

ThR transa double

of the wt rpoZ by Southern blot Sr. aureofuciens

compared

Microscopic

analysis

with the showed

long aerial hyphae without any sign of sporulation septa, a phenotype the same as that of St. coelicolor whiG mutant

(Chater,

1972), confirming

the functional

homol-

colony

mutants

Trends

Genet.

of Streptomyces

Microbial.

72

differentiation.

5 (1989) 372-377.

Kormanec,

J., Reiuchova,

B. and Farkasovsky, M.: Optimization of transformation and disruption of the h&l a homologue of the principal sigma factors. J. Gen.

aureofuciens

gene encoding

139 (1993) 2525-2529.

Lonetto, M.. Gribskov, M. and Gross, C.A.: The 0” family: sequence conservation and evolutionary relationships. J. Bacterial. 174 (1992) 3843-3849.

ACKNOWLEDGEMENTS

like to thank

J. Gen.

of B. suhtih. Cell 59 (1989) 133-143. Helmann, J.D., Marquez, L.M. and Chamberlin, M.J.: Cloning, sequencing and disruption of the Bacillus suhtih d8 gene. J. Bacterial. 170 (1988) 1568-1574.

Microbial.

We would

regulation

coelidor.

Chater, K.F., Bruton, C.J., Plaskitt, K.A., Buttner, M.J., Mendez. C. and Helmann, J.D.: The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility cr factor

Streptomyces

ogy of the whiG and rpoZ genes.

of Streptomyces

(1972) 9-28. Chater, K.F.: Multilevel

Mrs. Renata

Knirschova

for

excellent technical assistance, and Dr. Stefan Kuiela for critical reading of the manuscript. This work was supported in part by the Slovak Grant Agency for Science (grant No. GA999135).

Maxam, A.M. and Gilbert, W.: Sequencing end-labelled DNA with base specific chemical cleavages. Methods Enzymol. 65 ( 1980) 449-560. Ohnishi, K., Kutsukake, K., Suzuki, H. and Iino, T.: Gene,PiA encodes an alternative

sigma factor specific for flagella operons in Salmonellfi Mol. Gen. Genet. 221 (1990) 1399147. Soliveri, J., Vijgenboom, E., Granozzi, C., Plaskitt, K.A. and Chater, K.F.: Functional and evolutionary implications of a survey of varityphimurium.

ous actinomycetes for homologues of two Streptomyces coekolor sporulation genes. J. Gen. Microbial. 139 (1993) 2569-2578. Starnbach.

M.N. and Lory, S.: The ,fliA (rpoF) gene of Pseudomonas encodes an alternative sigma factor required for flagellin synthesis. Mol. Microbial. 6 (1992) 4599469.

REFERENCES

aeruginosu

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Tan, H. and Chater, K.F.: Two developmentally controlled promoters of Streptomyes coelicolor A3(2) that resemble the major class of motility-related

mapping

study

of white

(1993) 9333940.

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in other

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175