Nodulation and host‐specific recognition of legumes such as peas and Vicia spp. are encoded by the nodulation (nod) genes of Rhizobium leguminosarum biovar viciae. One of these genes, nodO, has been shown to encode an exported protein that contains a multiple tandem repeat of a nine amino acid domain. This domain was found to be homologous to repeated sequences in a group of bacterial exported proteins that includes haemolysin, cyclolysin, leukotoxin and two proteases. These proteins are secreted by a mechanism that does not involve an N‐terminal signal peptide. The NodO protein is present in the growth medium of Rhizobium bacteria induced for nod gene expression, and partial protein sequencing of the purified protein showed that there is no N‐terminal cleavage of the exported protein. It has been suggested that the internally repeated domain of haemolysin may be involved in Ca2(+)‐mediated binding to erythrocytes and we show that the NodO protein can bind 45Ca2+. It is proposed that the NodO protein may interact directly with plant root cells in a Ca2(+)‐dependent way, thereby mediating an early stage in the recognition that occurs between Rhizobium and its host legume.

A group of four rhi (rhizosphere-expressed) genes from the symbiotic plasmid of Rhizobium leguminosarum biovar viciae has been characterized. Although mutation of the rhi genes does not normally affect nodulation, in the absence of the closely linked nodulation genes nodFEL, mutations in the rhi genes can influence the nodulation of the vetch Vicia hirsuta. The DNA sequence of the rhi gene region reveals four large open reading frames, three of them constituting an operon (rhiL4BC) transcribed convergently toward the fourth gene, rhiR. rhL4BC are under the positive control of RhiR, the expression ofwhich is repressed by flavonoids that normally induce nod gene expression. This repression, which requires the nodD gene product (the transcriptional activator of nod gene expression), may be due to a cis effect caused by a high level of NodD-dependent expression from the adjacent nodO promoter, which is transcribed divergently from rhiR. RhiR shows significant similarities to a subfamily of transcriptional regulators that includes the LuxR and UvrC-28K proteins. RhiA shows limited homology to a short domain of the lactose permease, LacY, close to a region thought to be involved in substrate binding. No strong homologies were found for the other rhi gene products. It appears that RhiA and RhiB are cytoplasmic, whereas RhiC is a periplasmic protein, since it has a typical N-terminal transit sequence and a rhiC-phoA protein fusion expresses alkaline phosphatase activity. The biochemical role of the rhi genes has not been established, but it appears that they may play a role in the plant-microbe interaction, possibly by allowing the bacteria to metabolize a plant-made metabolite.There are many bacterial genes involved in the interaction between rhizobia and their legume hosts. In Rhizobium spp. many of these genes are present on large symbiotic plasmids. Initially, these genes were identified by isolating mutants unable to fix nitrogen or form nodules, but several nodulation (nod) genes in which mutations have little or no effect on nodulation have now been identified. Currently, over 30 different nod genes have been identified among a wide variety of rhizobia (32), and in general they are under the control of positively acting transcriptional regulators encoded by nodD genes. Several of the nod gene products are involved in the biosynthesis of low-molecular-weight signalling molecules that are specifically recognized by legumes. It is now clear that different rhizobia make different but related signalling molecules which are substituted glycolipids consisting of acylated oligoglucosamine signal molecules (43,51). The biosynthesis of these nodulation factors involves most of the nod gene products; different substitutions of the glycolipid, such as the presence of sulfate or the type of acyl group, are mediated by nod gene products and determine host specificity in the interaction between the bacterium and legume (43,51).In addition to the various nod genes and genes involved in nitrogen fixation, several other symbiotic-...

The rhi genes of Rhizobium leguminosarumbiovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372–376, 1996) therhiABC operon had been shown to be regulated by RhiR and to be induced by addedN-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3OH,C14:1-HSL). Mutagenesis of a cosmid carrying the rhiABC and rhiR gene region identified a gene (rhiI) that affects the level of rhiAexpression. Mutation of rhiI slightly increased the number of nodules formed on the pea. The rhiI gene is (likerhiA) regulated by rhiR in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of N-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces N-(hexanoyl)-l-homoserine lactone (C6-HSL) andN-(octanoyl)-l-homoserine lactone (C8-HSL). Both of these AHLs induced rhiA-lacZand rhiI-lacZ expression on plasmids introduced into anAgrobacterium strain that produces no AHLs, showing thatrhiI is positively regulated by autoinduction. However, in this system no induction of rhiA or rhiI with 3OH,C14:1-HSL was observed. Analysis of the spent culture supernatant of the wild-type R. leguminosarum bv. viciae revealed that at least seven different AHLs are made. Mutation ofrhiI decreased the amounts of C6-HSL and C8-HSL but did not block their formation, and in this background the rhiI mutation did not significantly affect the expression levels of the rhiI gene orrhiABC genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in R. leguminosarum bv. viciae and that they affect rhiI and rhiABC expression. We postulate that the previously observed induction of rhiA by 3OH,C14:1-HSL may be due to an indirect effect caused by induction of other AHL production loci.

Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and secretome are sharply distinct, such that a signal peptide-agnostic machine learning tool distinguishes cytoplasmic from secreted proteins with 95.5% success. Cytoplasmic polypeptides are enriched in aliphatic, aromatic, charged and hydrophobic residues, unique folds and higher early folding propensities. Secretory polypeptides are enriched in polar/small amino acids, β folds, have higher backbone dynamics, higher disorder and contact order and are more often intrinsically disordered. These non-random distributions and experimental evidence imply that evolutionary pressure selected enhanced secretome flexibility, slow folding and looser structures, placing the secretome in a distinct protein class. These adaptations protect the secretome from premature folding during its cytoplasmic transit, optimize its lipid bilayer crossing and allowed it to acquire cell envelope specific chemistries. The latter may favor promiscuous multi-ligand binding, sensing of stress and cell envelope structure changes. In conclusion, enhanced flexibility, slow folding, looser structures and unique folds differentiate the secretome from the cytoplasmome. These findings have wide implications on the structural diversity and evolution of modern proteomes and the protein folding problem.