TABLE 2.

Specific primers designed for PCR amplification and driven cloning strategy

Primer	Sequence (5′-3′)a	DNAb template	Cloning strategy	Recombinant plasmid
O-hmgA5 O-hmgA3	GGGGAGCTCGCTTTGGCCATGGGAGGGGAGGTT CAAGGTACCGGCGGCGAACCGTTCACTTC	KT2440	The 3,396-bp PhmgABC fragment that was PCR generated was double digested with SacI/KpnI and cloned into pUC18	pU-HMG
O-hpd5	TCCGGATCCCACGCAATCAATAATTTTCGC	KT2440	The 1,134-bp hpd gene that was PCR generated was double digested with KpnI/BamHI and cloned into pUC18	pU-hpd
O-hpd3	ACGGTACTCTAGAGGCGATCAGTCTGTGC		The 819-bp hpd internal fragment that was PCR generated was double digested with EcoRI/SalI and cloned into pK18mob	pK-dhpd
O-tyrB5 O-tyrB3	GGCTCTAGACTGTACGCCCTATCACACTC CCCTCTAGAAGTCAACCCACCCCCCAG	KT2440	The 1,278-bp tyrB1 gene that was PCR generated was digested with XbaI and cloned into pUC18	pU-tyrBI
O-hmgR5E O-hmgR3	GATGAATTCGGAACCTCCCCTC TATGTCGACTTCTCTCAGGAACTGC	KT2440	The 866-bp hmgR gene that was PCR generated was double digested with EcoRI/SalI and cloned into pQE32	pQ-hmgR
O-dphhA5 O-dphhA3	AGAGCATGCGGTGTGGAACACCC CCTGGATCCGGTCGAAGGCCTGG	KT2440	Cloning into pK18mob of the PCR-generated fragment digested with SphI/BamHI that contained a 539-bp internal sequence of phhA	pK-dphhA
O-dhmgA5 O-dhmgA3	CTGTCGACGAGCCCGCAGCCGATTCCT ACGGTACCTGTTCTCGGCCACCA	KT2440	The 677-bp hmgA internal fragment that was PCR-generated was double digested with SalI/KpnI and cloned into pK18mob	pK-dhmgA
O-dhmgR5 O-dhmgR3	GGCGTCGACGCATCCACCCCCGGCATCAGC GGGGTACCCGAACAGGATGCGGCCACCACC	KT2440	The 428-bp hmgR internal fragment that was PCR generated was double digested with SalI/KpnI and cloned into pK18mob	pK-dhmgR
O-Phmg5 O-Phmg3	GTGGTACCGACGTCGAGGGTGAGAGT GGGGATCCGGGGCCTTCTGCGGGGAGTTCTGC	KT2440	The 335-bp hmgR-hmgA intergenic region that was PCR generated was double digested with KpnI/BamHI and cloned into pSJ3	pSJ-Phmg-lacZ
HGA(5)2 HGA(3)8	CCGGCCGGCGTGAGCATCTACATCTACTG CTCGGCCACCATCCAGCGCGG	U	The 592-bp hmgA internal fragment that was PCR generated was cloned into pGEM-T easy and then digested with EcoRI and subcloned into pK18mob	pKhmgA
HGA(5)4 HGA(3)3	CCGCCCCGACAACCCGCTGCTAC GCGGCTGCGGGTCACCTTCGGG	U	The 433-bp hmgB internal fragment that was PCR generated was cloned into pGEM-T easy and then digested with EcoRI and subcloned into pK18mob	pKhmgB
HGA(5)6 HGA(3)5	GGCCTTGCGCACCGACGGTGG CCGATCCACTGGTTGACCTGCCCCTC	U	The 233-bp hmgC internal fragment that was PCR generated was cloned into pGEM-T easy and then digested with EcoRI and subcloned into pK18mob	pKhmgC
HPD(5)2	GCCGATGGAGCTGCGCCTGCCG	U	The 378-bp hpd internal fragment that was PCR generated was cloned into pGEM-T easy and then digested with EcoRI and subcloned into pK18mob	pKhpd
HPD(3)2	GAACTGCATCAGGAACTCTTCAATCTGGCC		The 378-bp hpd internal fragment that was PCR generated was cloned into pGEM-T easy and then digested with EcoRI and subcloned into pJQ200KS	pJQhpd
Phh(3)4 Phh(5)4	CTGGTGCTCAGGCTCGTCAGACAGACTGTAGAC CAACTGGGCGAGATCAACAAGGTGCTGGG	U	The 419-bp phhA internal fragment that was PCR generated was cloned into pGEM-T easy and then digested with EcoRI and subcloned into pK18mob	pKphhA
Hpd(5) Hpd(3)4	AATCAATAATTTTCGCTGCAGATGAG AGATCGTCCCCTCGCTGATGCTGGTGG	U	Cloning into pGEM-T easy of the PCR-generated fragment (1,245 bp) that contained the Hpd-coding region behind the lac promoter	pGhpd
HpdBam HpdPstB HpdPstX HpdXba	TCTTCGAGGATCCAATGGGCC TTTCAACCTGCAGGGCGCCCA GAGCGGCTGCAGGGTCACGG GGCGGCGCTATCTAGAGGGTATCAGTCGGTGC	U	The PCR-amplified 5′ end (primers HpdBam and HpdPstB) and 3′ end (primers HpdPstX and HpdXba) of the hpd gene were cloned in pJQ200KS as BamHI/PstI and PstI/XbaI fragments, respectively	pJQhpdBX
HmgBam HmgPstB HmgPstX HmgXba	CTACCTCAGCGGATCCGGCAAC GAAGAACACTCGCTGCAGGGAACGG TTTATCCACAGCCTGCAGTGC GGATGCGCGGGAAGCTCTAGAGG	U	The PCR-amplified 5′ end of hmgA (primers HmgBam and HmgPstB) and 3′ end of hmgC (primers HmgPstX and HmgXba) were digested with BamHI/PstI and PstI/XbaI respectively, and cloned into pJQ200KS to delete the hmg operon in P. putida U	pJQhmgBX
HmgA(5) HGA(3)6	GCCAGCAACTAGTCAGTCAGAGCCCGGAGG GGCAAGCTTCCGGCGGCGAACC	U	The PCR-amplified fragment (3,306 bp) containing the hmgABC genes was cloned into pBBR1MCS-3	pMChmg

^aEngineered restriction sites are underlined.

^bEach PCR was carried out with the two primers indicated and the genomic DNA of P. putida KT2440 or P. putida U as the template.