* Office of theCassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (PtaC) and the trpA terminator ( T , ) and then cloned between Not1 sites to construct the CAS-Nm (Ptac-npt//-TtpA) and CAS-Gm ( Pt , CP, , , c/ -aacC/ -T, )cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncPl plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a Not1 fragment into the Not1 site of a PUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-npt//-TtwA) and mTn5-Gm (containing P, CP, , , , -aacC/ -TtwA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-P,,-npt / / -T, ) and mTn5-GGm (containing gusA-Pt,~P,,,-dacC/-TtpA) can be used for transcription signal localization or insertional inactivation. The TAC-31 R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid-or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other Gramnegative bacteria.CAS-G Nm (gUSA-Pt,,-npt//-TtpA) or CAS-GGm (gUSA-Pt,CP,,,c/-da~CI-TtpA)

No abstract

An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by TnS mutagenesis, fails to grow below pH 6 9 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking TnS in Murdoch, Western Australia 61 50, Australia TG5-46 contains two open reading frames, ORFl (designated acts) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into acts in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (Acts-) and TG5-46 (ActR-), while fragments containing only actR or acts complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complemented not only TG5-46 but also RT295. Cloning DNA upstream from actR and acts into a broad-host-range lac2 expression vector and measuring bgalactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria.

2059Mutants of Rhizobium leguminosarum 300 which were unable to utilize one or more organic acids as growth substrates were obtained by Tn5 mutagenesis. Mutant strain MNF3080 was defective in dicarboxylate transport and was unable to grow on succinate. Strain MNF3085 was defective in phosphoenolpyruvate carboxykinase and hence could not carry out gluconeogenesis. This strain did not grow on pyruvate, succinate, glutamate or arabinose but grew on glucose and on glycerol. Strain MNF3075 was unable to utilize pyruvate; the biochemical lesion in this mutant was not identified. MNF3085 and MNF3075 were symbiotically effective. MNF3080 nodulated peas, but the nodules were ineffective in N2 fixation and displayed morphological abnormalities. These data support previous findings which suggest that utilization of exogenous dicarboxylates is essential for effective nodule development by R. leguminosarum.

Summary Two ‘calcium‐irreparable’ acid‐sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P‐type ATPase that belongs to the CPx heavy metal‐transporting subfamily. Here, we show that both actP‐knockout mutants show sensitivity to copper; omission of this heavy metal from low pH‐buffered media restores acid tolerance to these strains. Furthermore, complementation of the mutant phenotype requires only the actP gene. An actP–gusA fusion in R. leguminosarum was transcriptionally regulated by copper in a pH‐dependent manner. Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal‐responsive regulator (HmrR) that belongs to the merR class of regulatory genes. Insertional inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence. These observations suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.