Bacteroids of determinate nodules, in contrast to those found in indeterminate nodules, can accumulate up to 50% of their cellular dry mass as PHB (reviewed in [4]). The synthesis of PHB during symbiosis however, presumably occurs at the expense of symbiotic nitrogen fixation; a theory that is corroborated by the

observation that a phaC mutant of R. etli demonstrates higher levels of nitrogenase activity relative to wild-type [42]. Bacteroids PF-2341066 of indeterminate nodules do not accumulate PHB during symbiosis. It has been suggested [42] that this may be one of the reasons why the S. meliloti-alfalfa symbiosis is more effective than that of B. japonicum-soybean or R. etli-bean [43]. Interestingly the data presented in this paper suggest that forced accumulation of PHB by S. meliloti during symbiosis does not appear to have a negative effect on plant yield, suggesting that PHB synthesis during symbiosis is not the only determinant of symbiotic performance. Methods Bacterial strains, plasmids, growth VRT752271 clinical trial media and conditions All bacterial strains and plasmids used are listed in Table 5. Culture methods using Tryptone Yeast (TY), Luria Broth (LB), Yeast Mannitol Broth (YMB), Yeast Mannitol Agar (YMA), and Modified M9 medium supplemented with defined carbon sources, and antibiotic concentrations were carried out as described previously [23, 44]. Table 5 Bacterial Strains,

Plasmids and Phage Strain or Plasmid Relevant Characteristics Reference S. meliloti     Rm5000 SU47 rif5 [22] Rm1021 SU47 str-21, Sm R [50] Rm11105 Rm1021 phaC 1::Tn5 [23] Rm11107 Rm1021 bdhA1::Tn5 [23] Rm11144 Rm1021 phaC1::Tn5 -233 [23] Rm11347 Rm1021 phaB::ΩSmSp [24] Rm11417 Rm5000 phaZ::ΩSmSp This work Rm11430 Rm1021 phaZ::ΩSmSp This work Rm8369 Rm8002 exoF369::TnphoA [27] E. coli     DH5α F’ endA1 hsdR17 (r K m+) supE44 thi-1 recA1 gyrA Nal R relA1 Δ(lacIZYA-argF) U169 deoR (ϕ80dlac Δ(lacZ)M15) [51] MT607 pro-82 thi-1 hsdR17 supE44 recA56 [52] MT616 MT607 pRK600 [52] Plasmids     pK19mobsacB Suicide vector Km R [53] pGEMTEasy Cloning vector for PCR-generated DNA fragments,

Amp R Promega pAZ101 pGEMTeasy carrying 835 bp fragment of SMc02770 This work pAZ102 pAZ101 phaZ::OSmSp This work pAZ103 pK19mobsacB phaZ::ΩSmSp This work MK5108 supplier pRK7813 RK2 derivative carrying pUC9 polylinker. Tc R [54] pMA157 pRK7813 SMc02770 This work pD82 pLAFR1 cosmid clone from Rm1021 library carrying exoF and neighbouring Ribonucleotide reductase genes [26] pD82exoF::TnphoA pD82 exoF::TnphoA This work Phage     ϕM12 S. meliloti transducing phage [22] Genetics and molecular biology techniques Bacterial conjugations, ϕM12 transductions and homogenotizations were carried out as described previously [22]. DNA manipulations were performed using standard techniques [45]. DNA probes for Southern blot analyses were labelled with digoxygenin (DIG) using the DIG High-Prime Kit (Roche Diagnostics Canada) according to manufacturer’s instructions. Southern blots were performed using standard techniques [45].