The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.The symbiosis between legumes and the root nodule bacteria collectively known as rhizobia is of critical agronomic and environmental importance, accounting for the majority of the nitrogen fixed through biological processes. Rhizobia are phylogenetically diverse, falling into five genera of ␣-proteobacteria (Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, and Mesorhizobium) (64, 70) and at least two genera of -proteobacteria (Burkholderia and Ralstonia) (35). It is thought that the rhizobial lineages diverged well before the evolution of legumes and that the genes required for the formation of the symbiosis were subsequently acquired by lateral transfer from undefined sources (8,33). Reflecting the accessory nature of the traits, several species of rhizobia contain the genes required for nodulation and nitrogen fixation on large plasmids that can be cured under laboratory conditions without affecting the survival of the bacteria (31). Exceptions in which the symbiosis genes are encoded on the chromosome include Bradyrhizobium species, in which the symbiosis genes are clustered but not known to be mobile (18), and at least one strain of Mesorhizobium loti, strain ICMP3153, in which the genes are located on a mobile symbiosis island (57).M. loti is the microsymbiont of several Lotus species, including Lotus corniculatus and L. japonicus. The symbiosis island of M. loti strain ICMP3153 was discovered through its ability to transfe...
The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest.
The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N-and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (21). The resistance has historically been attributed to the presence in this organism of an outer membrane (OM) of low permeability (55), but it is increasingly clear that resistance owes much to the operation of broadly specific, so-called multidrug efflux systems (58-60, 64) that work synergistically with limited OM permeability (18,40,60). Several multidrug efflux systems in P. aeruginosa have been described to date (61), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (38). Hyperexpression of this system also occurs in so-called nalB (27, 28, 75, 88)-and nalC (75, 88)-type multidrug-resistant mutants. MexAB-OprM accommodates a broad range of structurally diverse antimicrobials, including dyes, detergents, inhibitors of fatty acid biosynthesis, organic solvents, disinfectants, and clinically relevant antibiotics (10,34,37,39,41,42,48,70,74,74,76), and is implicated in the export of homoserine lactones involved in quorum sensing (17, 57) and, possibly, virulence factors (22).The MexAB-OprM efflux system, like the other tripartite Mex efflux systems in P. aeruginosa, consists of an inner membrane (IM) drug-proton antiporter of the resistance-nodulation-cell division (RND) family (MexB), an OM channel-forming component (OprM; also called OM factor [OMF]), and a periplasmic membrane fusion protein (MFP) (MexA) (58, 86). Crystal structures have not yet been reported for any of the efflux components of P. aeruginosa, although structures are available for the homologous OM (TolC [35]) and RND (AcrB [52]) components of the Mex-like AcrAB-TolC multidrug efflux system of Escherichia coli. The TolC channel is a trimer and spans both the OM (as a -barrel) and periplasm (as a ␣-helical barrel) (35). Measuri...
Strains of Rhizobium leguminosarum bv. trifolii (Rlt) able to form effective nodules on Trifolium ambiguum (Caucasian clover, CC) form ineffective nodules on Trifolium repens (white clover, WC), whereas strains that form effective nodules on WC usually do not nodulate CC. Here, we investigate the genetic basis of the host-specific nitrogen-fixation phenotype of CC rhizobia. A cosmid library of the symbiotic plasmid from the WC rhizobium strain Rlt NZP514 was introduced into the CC rhizobium strain Rlt ICC105. An 18 kb Asp718 fragment containing the nifABHDKEN and fixABCX genes of NZP514 that imparted the Fix + phenotype was identified.Tn5 mutagenesis of this region revealed that the nifHDKEN, fixABC and nifB genes were required for the Fix + phenotype, but that the nifA gene was not. Introduction of several plasmids containing NZP514 nif/fix genes into an ICC105 nifA mutant strain demonstrated that the NifA protein of ICC105 was able to activate expression of the NZP514 nif/fix genes but not the ICC105 nif/fix genes in WC nodules. Reporter gene fusion studies showed that the host-specific regulation of the nif/fix genes depended on the DNA region between the promoters of the divergently transcribed nifH and fixA genes. We hypothesize that a protein acting either in response to a host-specific signal or in the absence of such a signal is able to bind upstream of the NifA-binding sites and interact with NifA to prevent it activating nif/fix gene expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.