TABLE 1.

Bacterial strains and plasmids used in this study

Strain, plasmid, or cosmid	Descriptiona	Reference or source
Bacillus sp. strain ATCC 27860	Indicator strain for alanyl clavam bioassay	34
K. pneumoniae ATCC 15380	Indicator strain for clavulanic acid bioassay	35
S. clavuligerus NRRL3585	Wild type	National Regional Research Laboratory (NRRL)
S. clavuligerus ΔccaR::tsrA	ccaR deletion mutant; ccaR replaced by tsr; Tsrr	2
S. clavuligerus C1G	Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the φC31 attB site in the chromosome	This study
S. clavuligerus C2G	Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the φC31 attB site in the chromosome	This study
Plasmids and cosmids
K6L2	pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster	1
p2.8-18	pUC18 containing the 5′ end of ceaS1 and upstream region	42
p5.7	pUC118 containing blsI, pahI, and flanking sequences	42
pBB5.3A	pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr	Jensen et al., unpublished
pCR2.1TOPO	E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr	Invitrogen
pIJ8660	Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr	41
pIJ8660-ceaS2	EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene	This study
pSET152	E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr	NRRL
pSET-ceaS1	EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene	This study
pTO6	pIJ8660 with aac(3)IV replaced by aadA; Spcr	28
pTO6-ceaS1	EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4	This study
pTOPO-cea52-8	pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region	This study
pTOPO-ceaS1-4	pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region	This study
pTZ40	pTZ18R containing an ∼26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr	Jensen et al., unpublished

^aAmp^r, ampicillin resistance; Apr^r, apramycin resistance; Kan^r, kanamycin resistance, Spc^r, spectinomycin resistance; Tsr^r, thiostrepton resistance.