Bacteriocins produced by different groups of bacteriums are ribosomally synthesized peptides or proteins with antimicrobic and specific counter bacterial interaction activity. Rhizobium leguminosarum is a Gram-negative dirt bacteria which plays an of import function in nitrogen arrested development in leguminose workss. Bacteriocins produced by different strains of R. leguminosarum are known to leave counter affects on other closely related strains. Recently a bacteriocin cistron has been isolated from R. leguminosarum bv. viceae strain LC-31. Our survey aims towards computational proteomic analysis and 3D structural mold of this fresh bacteriocin protein encoded by the above said cistron. Different bioinformatics tools and machine acquisition techniques were used for protein structural categorization. De novo protein mold was performed by utilizing I-TASSER waiter. The concluding theoretical account obtained was accessed by PROCHECK and DFIRE2, which confirmed that the concluding theoretical account is dependable. Until complete biochemical and structural informations of bacteriocin protein produced by R. leguminosarum bv. viceae strain LC-31 are determined by experimental agencies, this theoretical account can function as a valuable mention for qualifying this multifunctional protein.
Keywords: Bacteriocin ; Rhizobium ; Protein mold ; Nodulation ; Symbiosis ; Nitrogen arrested development
Bacteriocins are proteinaceous toxins secreted by Gram-positive and Gram-negative bacteriums. They have a narrow inhibitory spectrum against bacteriums that are closely related to the bring forthing bacteria. However, many of the bacteriocins produced by lactic acid bacteriums ( LAB ) have repressive spectra crossing beyond the genus degree and can potentially support unwanted microflora [ 13, 22, 28 ] . Bacteriocins were foremost identified about 100 old ages ago as a heat labile merchandise nowadays in civilizations of E. coli V and were toxic to E. coli S. These were given the name of colicin to place the bring forthing species [ 9 ] . Since so, bacteriocins have been found in all major line of descents of bacteriums and, more late, have been described as universally produced by some members of the Archaea [ 23-24 ] . Bacteriocins are normally ribosomally synthesized. The cistrons encoding bacteriocin production and unsusceptibility are organized in epichromosomal operon bunchs but some are besides chromosomally encoded, such as Lactobacillus sakei 5, which produces two chromosomally encoded bacteriocins [ 2, 19, 26 ] . These polypeptides have attracted much attending due to their possible usage as antibacterial agents for the intervention of infections, every bit good as saving of nutrient and animate being provender. The bacteriocin household includes a diverse figure of proteins in footings of size, microbic mark, manner of action, release, and unsusceptibility mechanisms, and can be divided into two chief groups: those produced by Gram-negative and those produced by Gram-positive bacteriums [ 8 ] .
The mutualism between leguminous plants and N2-fixing bacteriums ( Rhizobium ) is of immense agronomic benefit, leting many harvests to be grown without nitrogen-bearing fertilisers. It is a sophisticated illustration of coupled development between bacteriums and higher workss, climaxing in the organogenesis of root nodules [ 35 ] .
R. leguminosarum is a Gram-negative bacteria life in mutualism with leguminous workss in which it induces nitrogen-fixing root nodules [ 29 ] . These strains have been shown to bring forth bacteriocins that have been characterized as little, medium or big based on their assumed sizes and diffusion features. Large bacteriocins have been shown to resemble faulty bacteriophages [ 16, 20 ] . Small bacteriocins were found to be chloroform soluble and heat labile and to hold molecular multitudes of less than 2,000 Daltons [ 31 ] . Small bacteriocins were shown to be acylated homoserine lactone compounds related to quorum-sensing molecules [ 11, 27 ] . Very small is known about medium bacteriocins produced by R. leguminosarum. The ability of dirt bacteriums to bring forth bacteriocins, defined as specific, nonself-propagating inhibitory agents doing hostility between closely related strains, and bacteriocinogenic activity has been described in about all rhizobial species [ 30 ] . As bacteriocins act as polar substance in specific counter bacterial interaction, they can be potentially used to command bacterial works diseases by exercising their deadly effects on bacteriums of the same or related groups. Therefore bacteriocins have most of the belongingss considered desirable for microbic control [ 10, 23 ] . Subsequently on, it has been identified that rhizobial species are non merely involved in symbiotic N arrested development but besides exploit scope of mechanisms in direct or indirect mode to vie in nodulation and workss growing stimulation [ 12 ] . Despite of bacteriocins antibacterial activity the exact mechanism of their action is still mistily understood. However, protein theoretical accounts of bacteriocin can be created for the deeper penetrations into its construction and map. In the recent old ages, protein mold has become a promising tool with which we can foretell construction of those proteins which are usually hard to work out.
The purpose of this survey was to execute computational sequence analysis and 3D structural mold of a bacteriocin protein produced by R. leguminosarum bv. viciae strain LC-31. Understanding the bacteriocin 3D construction could assist us to understand how these extracellular proteins may lend to nodulation, suppression or suppression of other infective works bacteriums and related procedures that are known to be influenced by R.leguminosarum strains.
Material and Methods
Recently, isolation and word picture of the fresh bacteriocin cistron produced by R. leguminosarum bv. viceae strain LC-31 was performed [ 18 ] . Work performed by that group showed that the bacteriocin cistron has three constituents ; RzcA, RzcB and RzcD. While RzcB and RzcD are required for bacteriocin secernment, RzcA was found to really encode the bacteriocin protein. By utilizing recombination and cloning techniques, the nucleotide sequence of the RzcA fragment from R. leguminosarum bv. viceae strain LC-31 was determined to be 5′-TACGAAACTCTGGACGGCTCACCAATGCCGAAGCATCTCGTTGCCGA CGCATCACTTATTTATCGGCCCACCAATGCCACAT-3 ‘ .
In this survey, we have used this nucleotide sequence as a question for homology searching and computational mold of the bacteriocin protein from R. leguminosarum bv. viceae strain LC-31.
Protein sequence and construction analysis
Nucleotide sequence interlingual rendition
For the anticipation of structural belongingss and the 3D construction of any protein, we foremost necessitate its amino acid sequence. Up until now, the protein sequence of this specific bacteriocin cistron has non been uploaded to any database, therefore we used Translate [ 33 ] from Expasy to interpret the question nucleotide sequence into its protein sequence.
Primary and secondary constructions
ProtParam [ 33 ] was used to foretell physiochemical belongingss of the translated protein sequence. The parametric quantities computed by ProtParam include the molecular weight, theoretical pi, aminic acid composing, atomic composing, extinction coefficient, estimated half life, instability index, aliphatic index and expansive norm of hydropathicity ( GRAVY ) . Information sing the secondary construction of proteins allows us to foretell fold acknowledgment and Bachelor of Arts initio protein constructions, categorization of structural motives, and polish of sequence alliances. Secondary construction anticipations ( spiral, sheets, and spirals ) were made by utilizing different types of nervous webs. In comparing to other anticipation methods, machine larning attacks such as nervous webs have a major advantage, as these methods use developing sets of solved constructions to place common sequence motives associated with peculiar agreements of secondary constructions. The hierarchal nervous web ( HNN ) secondary construction anticipation method used in this survey is based on unreal nervous webs [ 4 ] . Two webs have been implemented in this plan ; these were the sequence to construction web and the construction to sequence web. JPred3 [ 3 ] is another secondary construction anticipation waiter that uses a dual nervous webs attack. The late updated Jnet algorithm provides a three-state ( ?-helix, ?-strand and spiral ) anticipation of secondary construction at an truth of 81.5 % . Another waiter used for secondary construction anticipations is PSIPRED [ 17 ] . It incorporates two feed-forward nervous webs which perform an analysis on end product obtained from Position Specific Iterated – BLAST ( PSI-BLAST ) . Using a really rigorous cross proof method to measure the method ‘s public presentation, PSIPRED achieves an mean truth of 80.7 % .
Subcellular localisation anticipation
Determining subcellular localisation is of import for understanding protein map and is a critical measure in genome note. PSORTb v3.0.2 [ 36 ] used here is the most precise bacterial localisation anticipation tool. It can do localisation anticipations for both Gram-positive and negative bacterial sequences and Archaea sequences.
3D structural mold and appraisal
The 3D construction is the concluding form that a functional protein assumes. Assorted adhering interactions between the side ironss on the amino acid residues determine the third construction of the protein. These interactions include salt Bridgess, disulfide bonds, hydrophobic interactions and H bonds. No high declaration X ray or NMR construction is available for the bacteriocin produced by R. leguminosarum bv. viceae strain LC-31. Therefore, we modeled the 3D construction utilizing two attacks: homology mold and de novo structural mold. Homology patterning plants best when the question matches an already present high declaration construction from the database with more than 60 % sequence similarity. In instances where no good templet is available, threading is done to foretell the 3D construction of the mark protein. For homology mold, we used an academic version of MODELLER v 9.2 [ 5 ] . In the instance of de novo structural mold, I-TASSER [ 25 ] was used. Furthermore, predicted 3D constructions were evaluated by PROCHECK [ 15 ] and DFIRE2 [ 34 ] and the computation of disulfide bond formation was checked by DiANNA [ 6 ] and DISULFIND [ 1 ] . Structures visual image was performed by UCSF Chimera 1.5 [ 21 ] .
Consequences and Discussion
Sequence interlingual rendition and homology seeking
The nucleotide sequence of R. leguminosarum bv. viceae strain LC-31 RzcA was obtained [ 18 ] and so subjected to nucleotide sequence interlingual rendition tools for finding of the bacteriocin protein sequence. A sum of six reading frames were generated. Stop codons were observed in all of the three 3′-5 ‘ reading frames ( informations non shown ) and they were discarded. The staying 5-3 ‘ frames which are given in table 1, were so subjected to blastp analysis for the intent of similarity searching, finding the degree of preservation among other bacteriocin proteins and finding of possible templets for 3D construction anticipation by homology mold. The hunt was performed against all non-redundant GenBank CDS interlingual renditions, PDB, SwissProt, PIR, and PRF databases utilizing default parametric quantities. A sum of 100 marks were obtained. However, the overall per centum of sequence homology was non satisfactory ( informations non shown ) . This explains the degree of diverseness that bacteriocin proteins have among different bacterial species and strains.
Primary and secondary construction analysis
ProtParam was used to analyse different belongingss of the translated reading frames. Frames 1 and 2 were found to be composed of 27 aminic acids whereas frame 3 had 26 aminic acids. The molecular weight for frame 1, 2 and 3 were calculated to be 2.96kDa, 3.11kDa, and 3kDa severally. Detailed physiochemical consequences for translated frames are given in table 2. The molecular weight and little protein length of bacteriocin produced by R. leguminosarum bv. viceae strain LC-31 suggests that it is biologically active and hence, may possess a broad scope of antimicrobic activity.
Different machine acquisition and nervous web based attacks were used to analyse the secondary constructions and predict the presence of alpha spirals, spirals, and extended strands for each frame. Prediction consequences from different tools are summarized in table 3. Overall, small fluctuation was observed in the consequences from different anticipation tools and waiters. Uniting the consequences from each attack, it was observed that reading frame 1 can organize two types of secondary constructions: alpha spirals and beta sheets. Reading frame 2 is predicted to hold merely beta sheets, whereas reading frame 3 can besides organize both alpha spirals and beta sheets. However, frame 3 was predicted to hold more secondary constructions as compared to border 1.
Subcellular localisation anticipations
Subcellular localisation is a cardinal functional property of a protein. Since cellular maps are frequently localized in specific compartments, foretelling the subcellular localisation of unknown proteins may be used to obtain utile information about their maps and to choose proteins for farther survey. Furthermore, analyzing the subcellular localisation of proteins is besides helpful in understanding disease mechanisms and for developing fresh drugs [ 32 ] . All bacterial proteins are synthesized in the cytol, and most remain at that place to transport out their alone maps. Other proteins, nevertheless, contain export signals that direct them to other cellular locations. In Gram-positive bacteriums, these include the cytoplasmatic membrane, cell wall and extracellular infinite, and in Gram-negative bacteriums, they include the cytoplasmatic membrane, the periplasm, the outer membrane and the extracellular infinite. In most instances, the whole protein is located in a individual compartment ; nevertheless, proteins can besides cross multiple localisation sites [ 7 ] . Bacterial cell surface and secreted proteins are of involvement for their possible as vaccinum campaigners or as diagnostic marks. It is besides known that bacteriocins are proteins secreted by bacteriums to kill other closely related bacterial species. We analyzed all three ( 5′-3 ‘ ) reading frames for their localisation potency by PSORTb. Based on anticipation consequences, reading frame 1 was found to be an unknown protein whereas, reading frames 2 and 3 were predicted to be extracellular proteins.
Third construction anticipation, rating and appraisal
Protein 3D constructions can supply us with precise information of how proteins interact and place in their stable conformation. Homology or comparative mold is one of the most common protein construction anticipation methods in structural genomics and proteomics. Therefore, we tried to pattern bacteriocin 3D construction utilizing homology mold. Numerous on-line waiters and tools are available for homology mold or comparative mold of proteins. Despite minimum alterations, one initial measure that was common in all mold tools and waiters was to happen the best duplicate templet. This was done by executing a sequence homology hunt by BLASTP. Templates are by experimentation determined 3D constructions of other proteins which portion certain degrees of sequence similarity with the question sequence. In the following measure, template sequence and the protein sequence whose construction has to be determined are aligned utilizing ClustalW2 [ 14 ] . A chiseled alliance is really of import for the anticipation of a dependable 3D construction. Swissmodel and Geno3D are two different waiters that were used to pattern 3D construction of bacteriocin. However, neither of these waiters was able to pattern the construction for any of the three reading frames, because of the absence of a suited templet. We were besides unable to pattern the 3D construction by MODELLER due to absence of any suited templet. These findings are in parallel to the above mentioned blast homology hunt consequences where the question does non portion more than 30 % individuality with any other protein in the protein databases at NCBI, PDB and Uniprot. Due to template dependent restrictions of homology mold, another computational biological science attack, known as de novo protein construction anticipation, was undertaken. Ab initio or de novo protein patterning plants on the rule that all the information for a protein construction lies in its amino acid sequence. This method builds a 3D construction based on physical rules instead than on antecedently solved constructions. Several on-line waiters, grid services and offline standalone package applications have been developed for de novo protein patterning. Amongst them, I-TASSER is one of the most widely used online waiters for protein construction and map anticipations. It works by utilizing a combination of ab initio folding and weaving methods. In this survey, I-TASSER was used for the anticipation of the bacteriocin 3D construction. Each reading frame was individually modeled in I-TASSER and five theoretical accounts were generated for each frame. Models generated for frames 1, 2 and 3 are shown in figure 1, 2 and 3 severally.
Once the theoretical accounts were generated, they were subjected to structural appraisal and proof utilizing PROCHECK, DFIRE2 and the C-Score values from the I-TASSER. Ramachandran secret plans were generated by PROCHECK. Additionally, the stereochemical qualities were assessed for each predicted theoretical account. Assessment consequences from PROCHECK are summarized in table 4. A sum of 15 structural theoretical accounts from three reading frames were analyzed in DFIRE2 and protein conformation free energy tonss were calculated. Free energy computations made by DFIRE2 are provided in table 4.
The concluding appraisal and proof decision of protein constructions were made on the footing of combined consequences from PROCHECK, DFIRE2 and I-TASSER ‘s C-Score. In the instance of frame 1, theoretical accounts 2 and 3 contained no residues in the disallowed part, one residue in the generously allowed part, and more than 57 % of residues were in the most favorite parts. By utilizing DFIRE2, predicted energy values for theoretical accounts 2 and 3 were found to be -23.55 and -22.48 severally, which are comparable to energy values of theoretical accounts 1, 4 and 5. For frame 2, hapless ramachandran secret plans were obtained. In the theoretical accounts generated for reading frame 3, theoretical accounts 1 and 3 had no residues in the disallowed part and one residue in the generously allowed part. However, theoretical account 5 besides had no residues in the disallowed part and merely one residue in the generously allowed part. The energy value for theoretical account 1 and 3 were calculated to be -30.64 and -27.64 severally which were the lowest among the five theoretical accounts. In add-on, C-Score value for theoretical account 1 ( -1.86 ) and 3 ( -1.91 ) were found to be highest among five theoretical accounts.
The presence of two or more than two cysteine residues consequences in formation of disulfide bonds which are known to play an of import function in bacteriocin protein stabilisation. Two cysteine residues were found in translated frame 3, one cysteine residue in frame 2 and no cysteine residue in frame 1. Therefore, reading frame 3 was inspected for possible disulfide adhering. Two waiters were used for the anticipation of disulfide adhering province and connectivity anticipation: DiANNA [ 6 ] and DISULFIND [ 1 ] . DiANNA employs a fresh diresidue nervous web based attack. In the initial phase, PSIPRED is run to foretell the protein ‘s secondary construction. PSIBLAST is so run against the non-redundant SwissProt database to obtain a multiple alliance of the input sequence. Following, the cysteine oxidization province is predicted, and so each brace of cysteines in the protein sequence is assigned a likeliness of organizing a disulfide bond. Finally, Rothberg ‘s execution of Gabow ‘s upper limit weighted duplicate algorithm is applied to diresidue nervous web tonss in order to bring forth the concluding connectivity anticipation. On the other manus, DISULFIND employs a support vector machines ( SVM ) binary classifier to foretell the bonding province of each cysteine, followed by a polish phase that classifies all the cysteines in a concatenation in a corporate manner. Almost similar consequences were obtained from both disulfide bonding and connectivity anticipation waiters. Two cysteine residues were found at places 14 and 25 in the reading frame 3, separated by a distance of 11 aminic acids. The presence of disulfide bond organizing cysteine residues is a characteristic characteristic of bacteriocins. It can besides be used as a footing for sub-grouping. It has been observed that the antibacterial efficiency of a bacteriocin increases with an addition in the figure of disulfide bonds. For illustration, pediocin AcH with two disulfide Bridgess has a wider scope of antimicrobic activity as compared to lactococcin B which has a individual disulfide span ( Ralph et al. , 1995 ) . Besides, disulfide bonds are known to be of import for the stableness of the bacteriocin protein ( Olivera et al. , 2003 ; Rober, 2005 ) . In understanding with the above mentioned construction appraisal analysis, frame 3 contains two cysteine residues with a extremely predicted potency for bond formation and may be a possible bacteriocin protein sequence.
Based upon the current cognition sing the activity and functionality of bacteriocins, and computational appraisal consequences, the lone theoretical accounts selected as representatives of bacteriocin 3D construction met the undermentioned standards: ( I ) predicted to be an extracellular protein with the maximal figure of secondary constructions in comparing to other predicted theoretical accounts ; ( two ) presence of cysteines residues for disulfide bonding ; ( three ) Ramachandran secret plans demoing the maximal figure of residues in allowed and the least figure of residues in disallowed parts ; and ( four ) lower limit free energy mark of protein conformation and highest value from C-Score. Therefore, we concluded that reading frame 1 is non likely to be the protein of the given bacteriocin, as it is non considered to be an extracellular protein by PSORTb, has less secondary construction anticipations than reading frame 3, and contains no cysteine residues. Reading frame 2 is least likely to be the protein of the given bacteriocin, as it is predicted to hold a lower degree secondary construction, which is required for bacteriocin map.
We propose that reading frame 3 is the coveted protein sequence of the bacteriocin in inquiry and theoretical accounts 1 and 3 are considered as the most likely 3D construction of the given bacteriocin. PSORTb predicted frame 3 to be an extracellular protein, with the maximal figure of secondary constructions compared to border 1 and 2. The presence of cysteine residues and disulfide bonding was confirmed by DiANNA and DISULPHID. PROCHECK, DFIRE2 and C-Score appraisals, supplying the best third constructions for frame 3. Although the adhering distance between the cysteine residues was found to be more than the allowed distance ( informations non shown ) , farther construction polishs of theoretical accounts 1 and 3 may ensue in the reduced distance between two cysteine residues.
With the aid of a chiseled construction of bacteriocin, one can foretell its functional and adhering sites, which can assist in understanding the multi-functional function of bacteriocin for competition in nodulation. This cognition can be farther used in drug design to heighten or stamp down the production of bacteriocin as required.
We are grateful to the Director of the National Centre of Excellence in Molecular Biology ( CEMB ) for supplying installations and our co-workers in Virology research lab, Functional Genomics research lab, and in Bioinformatics research lab ( CEMB ) for encouragement and support to transport out this work.
Conflicts of Interest
The writers declare that they have no struggle of involvement.