Regulation of pristinamycin biosynthesis

in S. pristinaespiralisJ. Guezguez, Y. Mast, E. Schinko and W. WohllebenUniversity of Tübingen, interfaculty Institute of Microbiology and Infection Medicine, Dpt. Microbiology / Biotechnology, Auf der Morgenstelle 28, 72076 Tübingen, Germany.

pristinamycin IA: R = Mepristinamycin IB: R = H

Fig. 1: Structure of pristinamycin I (I) and pristinamycin II (II).

pristinamycin IIA: dehydroprolinepristinamycin IIB: proline

II

The streptogramin antibiotic pristinamycin, produced by Streptomyces pristinaespiralis, is a mixture of two types of chemically unrelated compounds: pristinamycin PI and PII, which are produced in a ratio of 30:70. Pristinamycin PI is a cyclic hexadepsipeptide, belonging to the B-group of streptogramins, while pristinamycin PII has the structure of a polyunsaturated macrolactone of the A-group of streptogramins (Fig. 1).

PRISTINAMYCIN

Fig. 2: Schematic presentation of the mode of action of PI and PII.

Both compounds alone inhibit the protein biosynthesis by binding to the peptidyl transferase domain of the 50S subunit of the ribosome and are bacteriostatic. The A-group prevents the binding of the aminoacyl-tRNA to the 50S subunit of the ribosome. In contrast, the B-group facilitates the release of the peptidyl-tRNA from the ribosome (Fig. 2). Together they show a strong synergistic bactericidal activity, which can reach 100 times of the separate components (Harms et al., 2004).

MODE OF ACTION

Table 1: List of pristinamycin genes and their function in pristinamycin biosynthesis.

Fig. 3: Organization of the pristinamycin biosynthetic gene region. The 70 kb – gap is schematically shown in broken lines.

PRISTINAMYCIN BIOSYNTHETIC GENE REGION

Literature:

Bamas-Jacques, N., Lorenzon, S., Lacroix, P., de Swetschin, C. and Crouzet, J. 1999. Cluster organization of the genes of Streptomyces pristinaespiralis involved in pristinamycin biosynthesis and resistance elucidated by pulsed-field gel electrophoresis. Journal of Applied Microbiology 87: 939-948.Folcher, M., Gaillard, H., Nguyen, L.T., Nguyen, K.T., Lacroix, P., Bamas-Jacques, N., Rinkel, M., and Thompson, C.J. 2001. Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase. J. Biol. Chem. 276: 44297-44306.Harms, J.M., Schlünzen, F., Fucini, P., Bartels, H. and Yonath, A. 2004. Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin. BMC Biology 2:4.

N

NH

N

OO

OO

OH

O

Me O

malonyl-CoA

malonyl-CoA

malonyl-CoA

malonyl-CoA

malonyl-CoA

malonyl-CoAglycine

isobutyryl-CoAproline serine

I

O

O

NH

NH

O

NH

O

N

N N

O

OOMe

Me

N

O

OH

NR

L-proline L-dimethylamino-phenylalanine

L-pipecolic acid

L-phenylglycine

L-hydroxy-picolinic acid

L-threonine

L-2-amino-butyric acid

B-group (PI)

stimulates release of peptidyl-tRNA

A-group (PII)

prevents binding of amino acyl-tRNA

REGULATION OF THE PRISTINAMYCIN BIOSYNTHESIS

Seven regulatory genes were identified within the 210 kb region: spbR, papR1, papR2, papR3, papR4, papR5 and papR6 . SpbR (S. pristinaespiralis butyrolactone-responsive transcriptional repressor) is a specific receptor protein for γ-butyrolactones and the global regulator of pristinamycin biosynthesis (Folcher et al., 2001). papR1, papR2 and papR4 encode proteins that are homologous to SARPs which are pathway-specific transcriptional activator proteins, whereas papR3 and papR5 code both for proteins that belong to the family of TetR repressors. papR6 encodes a protein belonging to the class of response regulators (Table 2). Analysis of ΔpapR1 and ΔpapR2 deletion mutants supported these results. Furthermore papR3-, papR4-, papR5- and papR6- apramycin insertion mutants were constructed and their phenotypes were investigated. The effect of each mutation on pristinamycin biosynthesis was analyzed by HPLC (Fig.4).

On the basis of bandshift experiments we were able to prove the global regulatory and γ-butyrolactone binding function of SpbR. Furthermore EMSA (Fig. 5) and RT-PCR experiments (Fig. 6) showed that PapR2 is a hierarchical superior regulatory protein for the transcription of papR1 and the direct activator of the pristinamycin structural genes, whereas PapR1 is a “helper” protein of PapR2. As another SARP homologue, PapR4 could be a further activator of the pristinamycin structural genes . PapR5, as a TetR repressor protein, may temporarily retard the expression of papR1 and papR4 to ensure that the cells are able to gain self-resistance against pristinamycin. This repressing function of PapR5 could be abolished by the function of another TetR repressor, which might be the role of PapR3. PapR6 might control the transcription of papR4. Assuming the results of RT-PCR, bandshift and mutant analysis, a preliminary model of the regulation mechanism of pristinamycin biosynthesis was established (Fig. 7).

5‘ 3‘

50S

30S

A P

The pristinamycin biosynthetic gene cluster is partially characterized. It covers a region of about 210 kb where genes for PI and PII biosynthesis are interspersed (Fig. 3, table 1). Moreover, the pristinamycin coding region is interrupted by a cryptic secondary metabolite gene cluster which probably encodes for an actinorhodin-like compound.

Fig. 7 : Hypothetical regulation mechanism of pristinamycin biosynthesis.

Fig. 4: Results of the HPLC analysis of the S. pristinaespiralis wildtype, papR1-, papR2- deletion mutants and the papR3-, papR4-, papR5-, papR6- and spbR- apramycin insertion mutants. Pristinamycin PI and PII concentrations were followed over the time.

Table 2: List of pristinamycin regulatory genes and their deduced gene products.

Fig.5: EMSA: Binding of regulatory proteins PapR2, PapR4, PapR3 and PapR5 to promoter regions of papR1, papR3, papR4 and papR5. 1: Control, 2: Protein, 3: Protein + unspecific competitive DNA, 4: Protein + specific competitive DNA.

Regulator Function

SpbR Y-butyrolacton receptor

PapR1 SARP family regulator

PapR2 SARP family regulator

PapR3 TetR family repressor

PapR4 SARP family regulator

PapR5 TetR family repressor

PapR6 Response regulator

0 8 24 32 48 56 72 80 96 1040

0.0400000000000001

0.0800000000000001

0.12

0.16

0.2

WT∆spbR∆papR1∆papR2∆papR3∆papR4∆papR5∆papR6

PI

Time (h)

C (m

g/m

l)

0 8 24 32 48 56 72 80 96 1040

0.1

0.2

0.3

0.4

0.5

0.6

WT∆spbR∆papR1∆papR2∆papR3∆papR4∆papR5∆papR6

C (m

g/m

l)

PII

Time (h)

Pro-papR1

Pro-papR3

1 2 3 4

PapR5

1 2 3 4 1 2 3 4 1 2 3 4

Pro-papR1

Pro-papR2

Pro-papR4

Pro-papR5

Pro-papR1

Pro-papR4

PapR2 PapR4 PapR3

Fig. 6 : RT-PCR: Transcription of papR1, papR2 and papR4 in WT, ∆papR1, ∆papR2 and ∆papR4 at several time points under production conditions.

papR1

papR2

papR4

∆papR2∆papR1WT ∆papR4

24 48 72 96 24 48 72 96 24 48 72 96 24 48 72 96

papR5papR4papR3

papR1papR2

papR6

Pristinamycin Structural Genes

+

-

+

-

++

+ -

+

+

+

-

confirmed

suggested

Positive regulation

Positive regulation