Table 1 Plasmids and strains used in this study

pLAFR3.18

pLAFR vector containing the polycloning site of pTZ18R, Tc^R

[8]

pRAMM1

H.seropedicae NifA expressed from lac promoter

[26]

pK18mobsacBKm

Allelic exchange suicide vector; mobilized by E. coli S17-1 λpir, sacB,Km^R

[22]

pJQ200SK

Allelic exchange suicide vector; mobilized by E. coli S17-1, sacB,Gm^R

[23]

pRAM1T7

H.seropedicae NifA in pT7-7 vector

This study

pRAM1T7del

H.seropedicae NifA with a deletion of 576 bp

This study

pK18nifAdel

XbaI/BamHI fragment from pRAM1T7del

This study

pUC57Simple-3xFlag

5′-BamHI, KpnI, XhoI – 3xFlag-Stop – HindIII, SalI, XmaI-3′, Amp^R

Genscript® Corporation

pUC57nifAFlag

H. seropedicae nifA-3xFlag gene plus 647 bp of the downstream region, Amp^R

This study

pK18nifAFlag

H. seropedicae nifA-3xFlag gene plus 647 bp of the downstream region, Mob, SacB, Km^R

This study

pUC57fnr1Flag

H. seropedicae fnr1-3xFlag gene plus 1000 bp of the downstream region, Amp^R

This study

pJQfnr1Flag

H. seropedicae fnr1-3xFlag gene plus 1000 bp of the downstream region, Mob, SacB, Gm^R

This study

pPW452

lacZ fusion vector, Tc^R, Mob

[27]

pEMS140

Tc^R, Mob, nifB::lacZ fusion, nifB promoter cloned in pPW452

[6]

pRW1

Tc^R, Mob, nifA::lacZ fusion, nifA promoter cloned in pMP220

[20]

pRWC

pRW1, but with a 49 bp deletion including the UAS 2 site for NifA

[20]

pRW3

pRW1, but with a double mutation at the UAS 2 site for NifA (TGT ->TCT and ACA -> AGA)

[20]

pRW22A

pRW1, but with a single mutation at the −24/-12 promoter (G -> T at −25 residues)

[20]

Strains

Relevant characteristic

Source

SmR1

Herbaspirillum seropedicae Z78 but Sm^R 100 μg/mL, Nif+

[24]

MB1

Derived from SmR1 fnr1 deletion

[18]

MB2

Derived from SmR1, fnr2 deletion

[18]

MB3

Derived from SmR1, fnr3 deletion

[18]

MB13

Derived from MB1, fnr1 and fnr3 double deletion

[18]

MB21

Derived from MB2, fnr1 and fnr1 double deletion

[18]

MB23

Derived from MB2, fnr2 and fnr3 double deletion

[18]

MB231

Derived from MB23, fnr1, fnr2 and fnr3 triple deletion

[18]

MBN1

Derived from SmR1, but with 576 bp deletion in the nifA gene

This study

MBN2

Double fnr1, fnr3 deletion, plus a 576 bp deletion in the nifA gene

This study

MBN3

Triple fnr deletion, plus a 576 bp deletion in the nifA gene

This study

MBN4

Derived from SmR1, but with a C-terminal 3xFlag nifA gene

This study

MBN5

Double fnr1, fnr3 deletion, plus a C-terminal 3xFlag nifA gene

This study

MBN6

Triple fnr deletion, plus a C-terminal 3xFlag nifA gene

This study

MBF1

Derived from SmR1, but with a C-terminal 3xFlag fnr1 gene

This study