BacS: An Abundant Bacteroid Protein in Rhizobium etli Whose Expression Ex Planta Requires nifA

Abstract

Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption. This protein was not detected in bacteria cultured in tryptone-yeast extract. In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted. N-terminal sequencing of the protein led to isolation of a bacS DNA fragment. DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42. A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences. The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species. Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide. Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.

Characterization of the NifA-RpoN Regulon in Rhizobium etli in Free Life and in Symbiosis with Phaseolus vulgaris

“…Moreover, we confirmed the presence of the genes previously reported to be NifA-RpoN dependent in R. etli, including bacS, prxS, rpoN2, and iscN (16,31,43), and the presence of operons predicted in silico, including the iscN-nifUSW (16), nifHDKE1-yhd00103-yhd00102-fdxB, nifHDK3E2NX, and nifHD2-hemNd2-yhd00090 operons. For the last three operons, RNA transcripts that were the correct length were detected in Northern blots (data not shown).…”

“…However, NifAdependent gene activation occurs only at low oxygen concentrations (43,55). To date, only a few targets, including nifHDK, iscN nifUS, fixABC, prxS-rpoN2, melA, and bacS, have been reported to be members of the R. etli NifA-RpoN2 regulon during symbiosis with P. vulgaris (15,17,27,31,43,55).…”

The mosaic structure of the symbiotic plasmid of Rhizobium etliCFN42 and its relation to other symbiotic genome compartments

“…We predicted, as described in Materials and methods, potential RpoN-binding sites and UAS for NifA in the upstream region of several genes (see Additional data file 3). Both types of sites were also identified upstream of other genes; the reiterated yp003 , yp021 and yp099 genes that encode the recently described BacS protein, highly expressed in nodules [35]; yp010 in the putative operon for terpenoid synthesis [36]; the fixA , hesB , and cpxA5 genes; and yp104 , which encodes a toxin-transport-related protein. The expression of yp003 ( bacS ) and hesB ( iscN ) has recently been shown to depend on NifA [28,35].…”

“…There are 12 such families, with two or three members each (Figure 1). In addition to the nif family described above, five families are related to ERIS and the rest are various genes such as those that encode the BacS protein [35], or gene fragments.…”

A quinol oxidase, encoded by cyoABCD, is utilized to adapt to lower O2 concentrations in Rhizobium etli CFN42

“…The gel was stained with Coomassie blue ( Dzandu et al , 1984 ) to visualize protein amounts in each sample or the contents were electroblotted onto a nitrocellulose membrane. The blot was incubated with rabbit polyclonal antiserum against the BacS protein ( Jahn et al , 2003 ), and bound antibodies were detected with goat alkaline-phosphatase-conjugated anti-rabbit IgG (Sigma) that was developed with 5-bromo-4-chloro-3-indolyl phosphate.…”