An atlas of protein homo-oligomerization across domains of life.

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes.

De novo design of discrete, stable 310-helix peptide assemblies.

The α-helix is pre-eminent in structural biology1 and widely exploited in protein folding2, design3 and engineering4. Although other helical peptide conformations do exist near to the α-helical region of conformational space-namely, 310-helices and π-helices5-these occur much less frequently in protein structures. Less favourable internal energies and reduced tendencies to pack into higher-order structures mean that 310-helices rarely exceed six residues in length in natural proteins, and that they tend not to form normal supersecondary, tertiary or quaternary interactions. Here we show that despite their absence in nature, synthetic peptide assemblies can be built from 310-helices. We report the rational design, solution-phase characterization and an X-ray crystal structure for water-soluble bundles of 310-helices with consolidated hydrophobic cores. The design uses six-residue repeats informed by analysing 310-helical conformations in known protein structures, and incorporates α-aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilizing assemblies of 310-helices. This work provides principles and rules to open opportunities for designing into this hitherto unexplored region of protein-structure space.

Socket2: a program for locating, visualizing and analyzing coiled-coil interfaces in protein structures.

MOTIVATION: Protein-protein interactions are central to all biological processes. One frequently observed mode of such interactions is the α-helical coiled coil (CC). Thus, an ability to extract, visualize and analyze CC interfaces quickly and without expert guidance would facilitate a wide range of biological research. In 2001, we reported Socket, which locates and characterizes CCs in protein structures based on the knobs-into-holes (KIH) packing between helices in CCs. Since then, studies of natural and de novo designed CCs have boomed, and the number of CCs in the RCSB PDB has increased rapidly. Therefore, we have updated Socket and made it accessible to expert and nonexpert users alike. RESULTS: The original Socket only classified CCs with up to six helices. Here, we report Socket2, which rectifies this oversight to identify CCs with any number of helices, and KIH interfaces with any of the 20 proteinogenic residues or incorporating nonnatural amino acids. In addition, we have developed a new and easy-to-use web server with additional features. These include the use of NGL Viewer for instantly visualizing CCs, and tabs for viewing the sequence repeats, helix-packing angles and core-packing geometries of CCs identified and calculated by Socket2. AVAILABILITY AND IMPLEMENTATION: Socket2 has been tested on all modern browsers. It can be accessed freely at http://coiledcoils.chm.bris.ac.uk/socket2/home.html. The source code is distributed using an MIT licence and available to download under the Downloads tab of the Socket2 home page.

Bioconversion of lignin and its derivatives into polyhydroxyalkanoates: Challenges and opportunities.

Renewable energy resources are considered to be promising for the development of a sustainable circular economy. Among various alternatives, the microbial route for various biofuels production is quite lucrative. Use of cellulose and lignocellulose for methane, H2 , organic acids, ethanol, and cellulase has been explored a lot in the past few decades. The major leftover or a coproduct of these processes belongs to lignin-an aromatic cross-link polymer and one of the most abundant complex compounds on earth. A successful bioconversion route of lignin into high-value products is highly desirable for biorefinery perspective. It requires a complex set of enzymes/catalysts to decompose lignin through depolymerization and oxygen removal leading to its monomers that can be metabolized by engineered organisms to synthesize muconic acids, polyhydroxyalkanoates (PHAs), methane, and other high-value products. This article will focus on the opportunities and challenges in the bioconversion of lignin and its derivatives into PHAs.

Lipid production by Cryptococcus albidus using biowastes hydrolysed by indigenous microbes.

The efficiency of Cryptococcusalbidus was evaluated for its abilities to assimilate onion and apple hydrolysates as a medium for lipid production. Onion waste (OW) and apple waste (AW) were hydrolysed at an organic load of 2% total solids by indigenous microbes under mesophilic conditions. The indigenous microbes effectively hydrolysed both wastes giving the highest reducing sugar content of 4.8 g/L and 10.8 g/L with OW and AW hydrolysates, respectively. The microbiome analysis revealed that most of the indigenous microbes belonged to genus Bacillus and a significant population of α-proteobacteria and γ-proteobacteria were also present. Cell retention culture of C. albidus at a dilution rate of 0.01 h-1 resulted in a total dry cell weight (DCW) of 13.5 g/L with an intracellular lipid content of 20.0% at 168 h, corresponding to an enhancement of 3.48-folds and 2.37-folds in DCW and lipid concentration, respectively, as compared to batch fermentation.

RNAHelix: computational modeling of nucleic acid structures with Watson-Crick and non-canonical base pairs.

Comprehensive analyses of structural features of non-canonical base pairs within a nucleic acid double helix are limited by the availability of a small number of three dimensional structures. Therefore, a procedure for model building of double helices containing any given nucleotide sequence and base pairing information, either canonical or non-canonical, is seriously needed. Here we describe a program RNAHelix, which is an updated version of our widely used software, NUCGEN. The program can regenerate duplexes using the dinucleotide step and base pair orientation parameters for a given double helical DNA or RNA sequence with defined Watson-Crick or non-Watson-Crick base pairs. The original structure and the corresponding regenerated structure of double helices were found to be very close, as indicated by the small RMSD values between positions of the corresponding atoms. Structures of several usual and unusual double helices have been regenerated and compared with their original structures in terms of base pair RMSD, torsion angles and electrostatic potentials and very high agreements have been noted. RNAHelix can also be used to generate a structure with a sequence completely different from an experimentally determined one or to introduce single to multiple mutation, but with the same set of parameters and hence can also be an important tool in homology modeling and study of mutation induced structural changes.

Structural and functional analyses of PolyProline-II helices in globular proteins.

PolyProline-II (PPII) helices are defined as a continuous stretch of a protein chain in which the constituent residues have backbone torsion angle (φ, ψ) values of (-75°, 145°) and take up an extended left handed helical conformation, without any intra-chain hydrogen bonds. They are found to occur quite frequently in protein structures, with their number exceeding that of π-helices, though it is considerably less than that of α-helices and ß-strands. A relatively new procedure, ASSP, for the identification of regular secondary structures using Cα trace identifies 3597 PPII-helices in 3582 protein chains, solved at resolution ⩽2.0Å. Taking advantage of this significantly expanded database of PPII-helices, we have analyzed their structural and functional roles as well as determined the amino acid propensity within and around them. Though Pro residues are highly preferred, their presence is not a mandatory requirement for the formation of PPII-helices, since ∼40% PPII-helices were found to contain no Pro residues. Aromatic amino acids are avoided within this helix, while Gly, Asn and Asp residues are preferred in the proximal flanking regions. The PPII-helices range from 3 to 13 residues in length with the average twist and rise being -121.2°±9.2° and 3.0ű0.1Å respectively. A majority (∼72%) of PPII-helices were found to occur in conjunction with α-helices and ß-strands, and serve as linkers as well. The analysis of various intra-helical non-bonded interactions revealed frequent presence of CH⋯O H-bonds. PPII-helices participate in maintaining the three-dimensional structure of proteins and are important constituents of binding motifs involved in various biological functions.

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for Streptococcus.

The use of rrs (16S rRNA) gene is widely regarded as the "gold standard" for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4-7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

Searching Biomarkers in the Sequenced Genomes of Staphylococcus for their Rapid Identification.

Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5-6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.

Identification of local variations within secondary structures of proteins.

Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C(α) atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely α-helices, 310-helices, π-helices, extended ß-strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from α-helices and extended ß-strands, 310-helices and π-helices were also found to occur in substantial numbers. ASSP was able to discriminate non-α-helical segments from flanking α-helices, which were often identified as part of α-helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at http://nucleix.mbu.iisc.ernet.in/assp/index.php.

clpC operon regulates cell architecture and sporulation in Bacillus anthracis.

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

Genome Wide Search for Biomarkers to Diagnose Yersinia Infections.

Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4-6 base cutters). Yersinia species have 6-7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences-carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications.

Challenges and Opportunities for Customizing Polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) as an alternative to synthetic plastics have been gaining increasing attention. Being natural in their origin, PHAs are completely biodegradable and eco-friendly. However, consistent efforts to exploit this biopolymer over the last few decades have not been able to pull PHAs out of their nascent stage, inspite of being the favorite of the commercial world. The major limitations are: (1) the high production cost, which is due to the high cost of the feed and (2) poor thermal and mechanical properties of polyhydroxybutyrate (PHB), the most commonly produced PHAs. PHAs have the physicochemical properties which are quite comparable to petroleum based plastics, but PHB being homopolymers are quite brittle, less elastic and have thermal properties which are not suitable for processing them into sturdy products. These properties, including melting point (Tm), glass transition temperature (Tg), elastic modulus, tensile strength, elongation etc. can be improved by varying the monomeric composition and molecular weight. These enhanced characteristics can be achieved by modifications in the types of substrates, feeding strategies, culture conditions and/or genetic manipulations.

Genome Wide Analysis for Searching Novel Markers to Rapidly Identify Clostridium Strains.

Microbial classification is based largely on the 16S rRNA (rrs) gene sequence, which is conserved throughout the prokaryotic domain. The Ribosomal Database Project (RDP) has become a reference point for almost all practical purposes. The use of this gene is limited by the fact that it can be used to identify only to the extent to what has been known and is available in the RDP. In order to identify an organism whose rrs is not present in the RDP database, we need to generate novel markers to place the unknown on the evolutionary map. Here, sequenced genomes of 27 Clostridium strains belonging to 9 species have been used to identify two sets of genes: (1) common to most of the species, and (2) unique to a species. Combinations of genes (recN, dnaJ, secA, mutS, and/or grpE) and their unique restriction endonuclease digestion (AluI, BfaI and/or Tru9I) patterns have been established to rapidly identify Clostridium species. This strategy for identifying novel markers can be extended to all other organisms and diagnostic applications.

Genome Wide Analysis for Rapid Identification of Vibrio Species.

The highly conserved 16S rRNA (rrs) gene is generally used for bacterial identification. In organisms possessing multiple copies of rrs, high intra-genomic heterogeneity does not allow easy distinction among different species. In order to identify Vibrio species, a wide range of genes have been employed. There is an urgent requirement of a consensus gene, which can be used as biomarker for rapid identification. Eight sequenced genomes of Vibrio species were screened for selecting genes which were common among all the genomes. Out of 108 common genes, 24 genes of sizes varying from 0.11 to 3.94 kb were subjected to in silico digestion with 10 type II restriction endonucleases (RE). A few unique genes-dapF, fadA, hisD, ilvH, lpxC, recF, recR, rph and ruvB in combination with certain REs provided unique digestion patterns, which can be used as biomarkers. This protocol can be exploited for rapid diagnosis of Vibrio species.

Evolution of resistance to quorum-sensing inhibitors.

The major cause of mortality and morbidity in human beings is bacterial infection. Bacteria have developed resistance to most of the antibiotics primarily due to large-scale and "indiscriminate" usage. The need is to develop novel mechanisms to treat bacterial infections. The expression of pathogenicity during bacterial infections is mediated by a cell density-dependent phenomenon known as quorum sensing (QS). A wide array of QS systems (QSS) is operative in expressing the virulent behavior of bacterial pathogens. Each QSS may be mediated largely by a few major signals along with others produced in minuscule quantities. Efforts to target signal molecules and their receptors have proved effective in alleviating the virulent behavior of such pathogenic bacteria. These QS inhibitors (QSIs) have been reported to be effective in influencing the pathogenicity without affecting bacterial growth. However, evidence is accumulating that bacteria may develop resistance to QSIs. The big question is whether QSIs will meet the same fate as antibiotics.

Ecobiotechnological Approach for Exploiting the Abilities of Bacillus to Produce Co-polymer of Polyhydroxyalkanoate.

Ecobiotechnological approach is an attractive and economical strategy to enrich beneficial microbes on waste biomass for production of Polyhydroxyalkanoate (PHA). Here, six strains of Bacillus spp. were used to produce co-polymers of PHA from pea-shells. Of the 57 mixed bacterial cultures (BCs) screened, two of the BCs, designated as 5BC1 and 5BC2, each containing 5 strains could produce PHA co-polymer at the rate of 505-560 mg/l from feed consisting of pea-shell slurry (PSS, 2 % total solids) and 1 % glucose (w/v). Co-polymer production was enhanced from 65-560 mg/l on untreated PSS to 1,610-1,645 mg/l from PSS treated with defined hydrolytic bacteria and 1 % glucose. Supplementation of the PSS hydrolysate with sodium propionate enabled 5BC1 to produce co-polymer P(3HB-co-3HV) with a 3HV content up to 13 % and a concomitant 1.46-fold enhancement in PHA yield. Using the principles of ecobiotechnology, this is the first demonstration of PHA co-polymer production by defined co-cultures of Bacillus from biowaste as feed under non-axenic conditions.

Ecobiotechnological strategy to enhance efficiency of bioconversion of wastes into hydrogen and methane.

Vegetable wastes (VW) and food wastes (FW) are generated in large quantities by municipal markets, restaurants and hotels. Waste slurries (250 ml) in 300 ml BOD bottles, containing 3, 5 and 7 % total solids (TS) were hydrolyzed with bacterial mixtures composed of: Bacillus, Acinetobacter, Exiguobacterium, Pseudomonas, Stenotrophomonas and Sphingobacterium species. Each of these bacteria had high activities for the hydrolytic enzymes: amylase, protease and lipase. Hydrolysate of biowaste slurries were subjected to defined mixture of H2 producers and culture enriched for methanogens. The impact of hydrolysis of VW and FW was observed as 2.6- and 2.8-fold enhancement in H2 yield, respectively. Direct biomethanation of hydrolysates of VW and FW resulted in 3.0- and 1.15-fold improvement in CH4 yield, respectively. A positive effect of hydrolysis was also observed with biomethanation of effluent of H2 production stage, to the extent of 1.2- and 3.5-fold with FW and VW, respectively. The effective H2 yields were 17 and 85 l/kg TS fed, whereas effective CH4 yields were 61.7 and 63.3 l/kg TS fed, from VW and FW, respectively. This ecobiotechnological strategy can help to improve the conversion efficiency of biowastes to biofuels.

Biological machinery for the production of biosurfactant and their potential applications.

The growing biotechnology industry has focused a lot of attention on biosurfactants because of several advantages over synthetic surfactants. These benefits include worldwide public health, environmental sustainability, and the increasing demand from sectors for environmentally friendly products. Replacement with biosurfactants can reduce upto 8% lifetime CO2 emissions avoiding about 1.5 million tons of greenhouse gas released into the atmosphere. Therefore, the demand for biosurfactants has risen sharply occupying about 10% (∼10 million tons/year) of the world production of surfactants. Biosurfactants' distinct amphipathic structure, which is made up of both hydrophilic and hydrophobic components, enables these molecules to perform essential functions in emulsification, foam formation, detergency, and oil dispersion-all of which are highly valued characteristic in a variety of sectors. Today, a variety of biosurfactants are manufactured on a commercial scale for use in the food, petroleum, and agricultural industries, as well as the pharmaceutical and cosmetic industries. We provide a thorough analysis of the body of knowledge on microbial biosurfactants that has been gained over time in this research. We also discuss the benefits and obstacles that need to be overcome for the effective development and use of biosurfactants, as well as their present and future industrial uses.

Production of Polyhydroxyalkanoate Co-polymer by Bacillus thuringiensis.

Integrative processes for the production of bioenergy and biopolymers are gaining importance in recent years as alternatives to fossil fuels and synthetic plastics. In the present study, Bacillus thuringiensis strain EGU45 has been used to generate hydrogen (H2), polyhydroxybutyrate (PHB) and new co-polymers (NP). Under batch culture conditions with 250 ml synthetic media, B. thuringiensis EGU45 produced up to 0.58 mol H2/mol of glucose. Effluent from the H2 production stage was incubated under shaking conditions leading to the production of PHB up to 95 mg/l along with NP of levulinic acid up to 190 mg/l. A twofold to fourfold enhancement in PHB and up to 1.5 fold increase in NP yields was observed on synthetic medium (mixture of M-9+GM-2 medium in 1:1 ratio) containing at 1-2 % glucose concentration. The novelty of this work lies in developing modified physiological conditions, which induce bacterial culture to produce NP.