Copper is an essential transition metal for living organisms but it is detrimental in excess. The metalloregulatory protein copper‐sensing operon repressor (CsoR) in bacteria has evolved to prevent cytoplasmic copper toxicity. Cu(I)‐binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes but the mechanism is unknown. A phylogenetic analysis of 227 DUF156 protein members including biochemically or structurally characterized CsoR/RcnR repressors reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)‐bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2‐helix between two conserved copper‐ligating residues and folds an N‐terminal tail (residues 12‐19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo‐state with these dynamics quenched upon Cu(I) binding. Small angle X‐ray scattering (SAXS) experiments on an N‐terminally truncated Gt CsoR (∆2‐10) reveal that the Cu(I)‐bound tetramer is hydrodynamically more compact than is the apo‐state. A mutational analysis of residues critical to N‐terminal tail folding reveals that these residues function to stabilize the apoprotein‐DNA complex and/or control the extent of allosteric negative regulation of DNA binding by Cu(I), but to varying degrees. The mechanism of Cu(I)‐mediated allosteric switching in CsoRs is discussed. Grant Funding Source: Supported by NIH grant GM042569

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The ubiquitous Cation Diffusion Facilitator proteins (CDF) play a key role in maintaining the cellular homeostasis of essential metal ions. Previous neighbor-joining phylogenetic analysis classified CDF proteins into three substrate-defined groups: Zn(2+), Fe(2+)/Zn(2+) and Mn(2+). These studies were unable to discern substrate-defined clades for Ni(2+), Co(2+), Cd(2+) and Cu(2+) transporters, despite their existence in this family. In this study we improved the accuracy of this previous functional classification using a phylogenomic approach based on a thorough maximum-likelihood phylogeny and the inclusion of recently characterized CDF transporters. The inference of CDF protein function predicted novel clades for Zn(2+), Fe(2+), Cd(2+) and Mn(2+). The Ni(2+)/Co(2+) and Co(2+) substrate specificities of two clades containing uncharacterized proteins were defined through the functional characterization of nepA and cepA metal inducible genes which independently conferred Ni(2+) and Co(2+) resistances to Rhizobium etli CFN42 and increased, respectively, Ni(2+)/Co(2+) and Co(2+) resistances to Escherichia coli. Neither NepA nor CepA confer Zn(2+), Fe(2+) and Mn(2+) resistances. The ability of NepA to confer Ni(2+)/Co(2+) resistance is dependent on clade-specific residues Asn(88) and Arg(197) whose mutations produce a non-functional protein.

Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9-to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.Bacteria belonging to the genus Rhizobium are able to establish a symbiotic relationship with the roots of leguminous plants, in which the bacteria provide fixed nitrogen to the plants in exchange for a carbon source and a secure environment. Usually, the bacterial genetic information required for establishment of this symbiotic relationship is localized on symbiotic plasmids (pSyms) (16, 39).Self-transmissible pSyms have been described in Rhizobium leguminosarum (4, 24). A recent report indicated that transfer of one of these plasmids (pRL1JI) is regulated by a quorumsensing mechanism (29, 51). It has been shown that transfer genes localized on the pSym of Rhizobium sp. strain NGR234 are induced by quorum-sensing regulators, although the conjugal efficiency of this plasmid is extremely low (21). Also, self-transmissible cryptic plasmids have been described in Sinorhizobium meliloti (31). However, data regarding the transfer mechanism of these plasmids are scarce. Sequence analysis of various rhizobial symbiotic regions has revealed the presence of transfer-related genes and sequences homologous to the luxI-luxR type of quorum-sensing regulators (2,15,19,30). Nevertheless, the conditions unde...

The functional analysis of plasmids in Rhizobium strains has concentrated mainly on the symbiotic plasmid (pSym). However, genetic information relevant to both symbiotic and saprophytic Rhizobium life cycles, localized on other 'cryptic' replicons, has also been reported. Information is reviewed which concerns functional features encoded in plasmids other than the pSym: biosynthesis of cell surface polysaccharides, metabolic processes, the utilization of plant exudates, aromatic compounds and diverse sugars, and features involved symbiotic performance. In addition, factors which affect plasmid evolution through their influence on structural features of the plasmids, such as conjugative transfer and genomic rearrangements, is discussed. Based on the overall data, we propose that together the plasmids and the chromosome constitute a fully integrated genomic complex, entailing structural features as well as saprophytic and cellular functions.