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Symbiotic partnerships with rhizobial bacteria enable legumes to grow without nitrogen fertilizer because rhizobia convert atmospheric nitrogen gas into ammonia via nitrogenase. After Sinorhizobium meliloti penetrate the root nodules that they have elicited in Medicago truncatula, the plant produces a family of about 700 nodule cysteine-rich (NCR) peptides that guide the differentiation of endocytosed bacteria into nitrogen-fixing bacteroids. The sequences of the NCR peptides are related to the defensin class of antimicrobial peptides, but have been adapted to play symbiotic roles. Using a variety of spectroscopic, biophysical and biochemical techniques, we show here that the most extensively characterized NCR peptide, 24 amino acid NCR247, binds haem with nanomolar affinity. Bound haem molecules and their iron are initially made biologically inaccessible through the formation of hexamers (6 haem/6 NCR247) and then higher-order complexes. We present evidence that NCR247 is crucial for effective nitrogen-fixing symbiosis. We propose that by sequestering haem and its bound iron, NCR247 creates a physiological state of haem deprivation. This in turn induces an iron-starvation response in rhizobia that results in iron import, which itself is required for nitrogenase activity. Using the same methods as for L-NCR247, we show that the D-enantiomer of NCR247 can bind and sequester haem in an equivalent manner. The special abilities of NCR247 and its D-enantiomer to sequester haem suggest a broad range of potential applications related to human health.

The present world is data world; without this data cannot survive in present stage. This data produced more from social media; this media data is public data; This public data not have good security; so to overcome this issue we apply the Salsa method. This method easily hack the data from the hackers. RBJ32 method has 5 steps. 1. Applying the key and multiply that key; 2. To apply the prime number in the S 2 and T 2 ; 3. To calculate the EA1 and EA2; 4. To swap the EA1 and EA2 in matrix EnA; 5. Apply the column operations. The proposed method provides good security while comparing with Salsa method.

Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3' end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti Delta ybeY strain. We also showthat Delta ybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a Delta ybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria. National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM31030, P30 ES002109, 1R15AI117648]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM031030] National Institutes of Health (NIH) [GM31030 to G.C.W., P30 ES002109 to theMIT Center for Environmental Health Sciences and 1R15AI117648 to C.C.]; G.C.W. is an American Cancer Society Professor. Funding for open access charge: NIH [GM031030].

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria., National Institutes of Health (U.S.) (Grant GM31010), National Institutes of Health (U.S.) (Grant GM17151)

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda–sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda–β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda–β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda–β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function.

The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (β-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-β interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-β complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-β interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein.

The E. coli dnaN-encoded β sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of in vitro experiments indicate that the loading of β clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to E. coli viability, as well as the ability of the β clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of β clamp-DNA interactions to the ability of E. coli to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct β clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the β clamp through which the DNA template threads. Twenty of these 22 dnaN mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting hda function. Taken together, these results demonstrate that discrete β clamp-DNA interaction regions contribute to the ability of E. coli to tolerate specific classes of DNA damage.

The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.