FEMS Microbiology Letters 120 (1994) 187-190 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

187

FEMSLE 06054

Protein tyrosine phosphorylation in streptomycetes

Barbara Waters, Dusica Vujaklija, Michael R. Gold and Julian Davies *

Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, B.C., Canada V6T 1Z3

(Received 8 April 1994; revision received and accepted 4 May 1994)

Abstract: Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

Key words: Streptomyces spp.; Tyrosine phosphorylation; Secondary metabolism; Regulation of metabolism

Introduction

In eukaryotic cells, the control of proliferation and differentiation is mediated by multiple signal transduction pathways that are regulated by the action of tyrosine kinases and tyrosine phos- phatases. Although tyrosine kinase signalling is considered to be largely a eukaryotic process, the presence of tyrosine kinase activity and tyrosine phosphorylated proteins in bacteria has been re- ported [1,2]. In addition, it has been demon- strated recently that certain bacterial species (e.g. Yersinia sp., Salmonella sp. and enteropathogenic Escherichia coli) have evolved specific pathogenic

* Corresponding author. Tel.: (604) 822 2501; Fax: (604) 822 6041; e-mail: davies, avies.microbiology.ubc.ca

strategies that affect protein tyrosine phosphory- lation in the host [3].

Streptomycetes have a complex differentiation cycle that is genetically determined but which also responds to variations in the concentrations of low molecular mass components in the culture medium or cell cytoplasm [4]. Ishizuka et al. [5] reported the existence of regulatory genes in streptomycetes which resemble a typical prokary- otic two-component system. The products of these genes may be involved in a signal transduction network that regulates secondary metabolism. Several studies suggest a role for protein phos- phorylation in the regulation of metabolism in streptomycetes. For example, Stowe et al. [6] ex- amined the protein kinase activity in cell-free extracts from S. coelicolor and found that the number and extent of phosphorylated proteins

SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 2 0 0 - B

188

206

105 70.8

43.6

28.2

17.9

1 2 3 4 5 6

Fig. 1. Tyrosine phosphorylation in proteins from Strepto- rnyces species. Lane 1, S. lauendulae; lane 2, S. lividans; lane 3, S. griseus; lane 4, S. coelicolor; lane 5, S. rirnosus; lane 6, S. hygroscopicus. Extracts were prepared from cultures in loga- rithmic growth in minimal growth medium, and 10 p,g total protein was loaded per lane. The molecular masses of the

marker proteins are indicated (in kDa).

varied with the culture stage of the mycelia. In addition, Hong et al. [7] demonstrated that pro- tein kinase inhibitors known to be active against eukaryotic kinases block cellular differentiation and secondary metabolism of S. griseus. However, these studies did not identify the nature of the protein phosphorylation. We describe experi-

ments demonstrating that several different species of Streptornyces exhibit phosphorylation of tyro- sine residues in proteins during growth and that this modification is growth phase dependent.

Materials and Methods

Microorganisms and culture conditions The following bacterial strains were used in

this work: Streptomyces lividans TK23, S. coeli- color A3(2) [8], S. griseus ATCC 23345, S. hygro- scopicus HP5-29 [9], S. lavendulae ATCC 8664, and S. rimosus ATCC 10970. The liquid rich medium for S. hygroscopicus was prepared as described [9]. R2YE medium without agar, sup- plemented with 0.15% proline and 0.1% as- paragine, as well as a minimal medium [8] were used for culturing the bacterial strains mentioned above.

Preparation of cell-free extracts The mycelia were harvested from 100-ml cul-

tures at different stages of growth by centrifuga- tion at 5000 × g and washed with 10 mM Tris, 1 mM EDTA, 1 mM DTT and 10% glycerol. The wet cell biomass was determined, then the cells were suspended in the same buffer with phenyl- methylsulfonyl fluoride (0.5 mM) and pepstatin

206

105 70.8

43.6

28

h g I h g I h g I h g I

17.9

A B C D

Fig. 2. The 4G10 monoclonal antibody is specific for phosphotyrosine. Antibody binding to proteins in extracts from Streptomyces hygroscopicus (h), griseus (g) and lividans (1) (A) was blocked following incubation with 5 mM phosphotyrosine (B) but not by 5 mM phosphoserine (C) or phosphothreonine (D). Extracts were prepared from logarithmic growth phase cultures in minimal medium

and 10/zg total protein was loaded per lane.

(1.5 ~ M ) and d i s rup t ed by sonica t ion o r by ho- mogen i sa t i on with a glass b e a d mill; s imilar re- sults were o b t a i n e d with both . Cell debr i s was r emoved by cen t r i fuga t ion at 12000 x g and the s u p e r n a t a n t was used as a ce l l - f ree extract .

Gel electrophoresis and immunoblot t ing Pro te ins were reso lved by S D S - P A G E u n d e r

reduc ing condi t ions in 12% gels. Tyros ine -phos - pho ry l a t ed p ro t e ins were d e t e c t e d on W e s t e r n blots with the 4G10 monoc lona l an t i -phos- pho ty ros ine an t ibody (Ups t a t e Biologicals Inc., Lake Placid, NY). A n t i b o d y b ind ing was visu- a l i sed with p e r o x i d a s e - c o u p l e d goat an t i -mouse I g G (d i lu ted 1 : 10000) and chemi luminescen t de- t ec t ion (ECL; A m e r s h a m ) .

100

o° 10

0.1

A

5 4

i I i I i I i I

20 40 60 80 T i m e { h o u r s ' }

100 120

B

206 105

70.8

43.6

1 2 3 4 5 6

28.2

Fig. 3. (A) Growth of S. hygroscopicus in liquid rich medium. 100 ml cultures were initiated with a 2% (v/v) inoculum from a logarithmic phase culture and incubated with shaking at 30°C. The mycelia were harvested at the time points indicated in the figure, the wet cell biomass was determined and ex- tracts were prepared as described in Materials and Methods. (B) Protein tyrosine phosphorylation decreased in S. hygro- scopicus during growth in rich medium. Tyrosine phosphoryla- tion was compared in extracts prepared from cultures in logarithmic growth (lanes 1 and 2), late logarithmic growth (lane 3), early stationary (lane 4) and late stationary phase

(lanes 5, 6). 10/~.g total protein was loaded in each lane.

189

206

105 70.8

43.6

28

1 2 3 4 5 6 7 8 9

17.9

Fig. 4. Decreased tyrosine phosphorylation in S. lividans grown in R2YE medium during progression from logarithmic growth (lanes 1, 2) to early stationary (lane 3) and later stationary phase (lane 4). The pattern of phosphorylation seen in early exponential growth in minimal medium (lane 5) was similar to that in rich medium. However, in addition to decreased tyro- sine phosphorylation in late logarithmic (lane 6) and early stationary phase cells (lane 7), phosphorylation of different proteins was seen. Increased tyrosine phosphorylation was noted (arrow) in one S. lavendulae protein as the cells pro- gressed from logarithmic growth (lane 8) to stationary phase (lane 9) in minimal medium. 10/~g total protein was loaded in

each lane.

Results and Discussion

Extrac ts of severa l d i f fe ren t species of s t rep to- myce tes were p r e p a r e d and the p re sence of tyro- s i ne -phospho ry l a t ed p ro t e ins was ana lysed by im- munob lo t t i ng with a phospho ty ros ine - spec i f i c an- t ibody. Tyros ine phospho ry l a t i on of a wide range of p ro t e ins was obse rved in all s t ra ins tes ted . Each s t ra in exhib i ted a un ique p a t t e r n (Fig. 1) which may ref lec t d i f fe rences in s econda ry metabo l i sm. Iden t i ca l resul ts (not shown) were o b t a i n e d with a d i f fe ren t an t i -phospho ty ros ine monoc lona l ( IG2; B o e h r i n g e r - M a n n h e i m ) .

The specif ici ty of the an t ibody for phospho ty - ros ine was d e m o n s t r a t e d by showing tha t 5 m M phospho ty ros ine , bu t not 5 m M p h o s p h o s e r i n e or p h o s p h o t h r e o n i n e (Fig. 2) b locked the b ind ing of this an t ibody to S. hygroscopicus, S. griseus and S. lividans pro te ins tha t had been t r ans fe r r ed to n i t rocel lu lose . T h e except ion was a p ro t e in (molecu la r mass abou t 34 000 Da) which was rec- ognized by secondary an t ibody a lone (da t a not shown).

190

The pattern of protein tyrosine phosphoryla- tion in extracts of S. hygroscopicus changed dur- ing growth in rich medium (Fig. 3A, B). Protein tyrosine phosphorylation decreased as the ceils approached stationary phase. A similar decrease was seen in S. lividans (Fig. 4) and in S. lacendu- lae proteins (data not shown) following continued growth in rich (R2YE) medium. When the bacte- ria were grown in minimal medium, a decrease in overall tyrosine phosphorylation was observed during stationary phase (Fig. 4). However, in early stationary phase, phosphorylation of proteins was seen in S. lividans extracts and increased tyrosine phosphorylation (arrow) of one S. lavendulae pro- tein was noted along with decreased phosphoryla- tion in others. We have observed protein kinase activity in cell-free extracts of these strains (re- suits not shown), but the identities of these ki- nases, as well as their substrates, remain to be determined.

Streptomyces spp. possess a complex and tightly regulated growth cycle [10]. These results repre- sent the first demonstration that streptomycetes employ a type of covalent modification of pro- teins that is strongly correlated with signal trans- duction and growth regulation in eukaryotes. Growth-related changes in the levels of protein tyrosine phosphorylation suggest that post-trans- lational modification of this type may play a role in growth regulation in streptomycetes; late expo- nential and stationary growth phases represent significant shifts in metabolism, for example, to- wards sporulation or secondary metabolite forma- tion. Given that most (if not all) streptomycetes have a very complex network of secondary metabolite production, it seems possible that pro- tein tyrosine phosphorylation could play a role in shunting cellular metabolism from primary to sec- ondary metabolism (e.g. antibiotics). The pres- ence of tyrosine kinases in streptomycetes sug- gests that these bacteria are likely to possess cognate tyrosine phosphatases that regulate the levels of tyrosine phosphorylation. Furthermore,

it is possible that analogues of other eukaryotic signalling components associated with tyrosine phosphorylation will be found in streptomycetes.

Acknowledgements

We wish to thank the Medical Research Coun- cil of Canada and the Natural Sciences and Engi- neering Research Council for financial support of this work.

References

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