Investigation of singlet-oxygen-responsive genes in the cyanobacterium Synechocystis PCC 6803.

Singlet oxygen (1O2) is an important reactive oxygen species whose formation by the type-II, light-dependent, photodynamic reaction is inevitable during photosynthetic processes. In the last decades, the recognition that 1O2 is not only a damaging agent, but can also affect gene expression and participates in signal transduction pathways has received increasing attention. However, contrary to several other taxa, 1O2-responsive genes have not been identified in the important cyanobacterial model organism Synechocystis PCC 6803. By using global transcript analysis we have identified a large set of Synechocystis genes, whose transcript levels were either enhanced or repressed in the presence of 1O2. Characteristic 1O2 responses were observed in several light-inducible genes of Synechocystis, especially in the hli (or scp) family encoding HLIP/SCP proteins involved in photoprotection. Other important 1O2-induced genes include components of the Photosystem II repair machinery (psbA2 and ftsH2, ftsH3), iron homeostasis genes isiA and idiA, the group 2 sigma factor sigD, some components of the transcriptomes induced by salt-, hyperosmotic and cold-stress, as well as several genes of unknown function. The most pronounced 1O2-induced upregulation was observed for the hliB and the co-transcribed lilA genes, whose deletion induced enhanced sensitivity against 1O2-mediated light damage. A bioreporter Synechocystis strain was created by fusing the hliB promoter to the bacterial luciferase (lux), which showed its utility for continuous monitoring of 1O2 concentrations inside the cell.

Intrabacterial lipid inclusion-associated proteins: a core machinery conserved from saprophyte Actinobacteria to the human pathogen Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the aetiologic agent of tuberculosis (TB), stores triacylglycerol (TAG) in the form of intrabacterial lipid inclusions (ILI) to survive and chronically persist within its host. These highly energetic molecules represent a major source of carbon to support bacterial persistence and reactivation, thus playing a leading role in TB pathogenesis. However, despite its physiological and clinical relevance, ILI metabolism in Mtb remains poorly understood. Recent discoveries have suggested that several ILI-associated proteins might be widely conserved across TAG-producing prokaryotes, but still very little is known regarding the nature and the biological functions of these proteins. Herein, we performed an in silico analysis of three independent ILI-associated proteomes previously reported to computationally define a potential core ILI-associated proteome, referred to as ILIome. Our investigation revealed the presence of 70 orthologous proteins that were strictly conserved, thereby defining a minimal ILIome core. We further narrowed our analysis to proteins involved in lipid metabolism and discuss here their putative biological functions, along with their molecular interactions and dynamics at the surface of these bacterial organelles. We also highlight the experimental limitations of the original proteomic investigations and of the present bioinformatic analysis, while describing new technological approaches and presenting biological perspectives in the field. The in silico investigation presented here aims at providing useful datasets that could constitute a scientific resource of broad interest for the mycobacterial community, with the ultimate goal of enlightening ILI metabolism in prokaryotes with a special emphasis on Mtb pathogenesis.

Design, synthesis and antibacterial activity against pathogenic mycobacteria of conjugated hydroxamic acids, hydrazides and O-alkyl/O-acyl protected hydroxamic derivatives.

With the aim to discover new antituberculous molecules, three novel series of 23 hydroxamic acids, 13 hydrazides, and 9O-alkyl/O-acyl protected hydroxamic acid derivatives have been synthesized, and fully characterized by spectral 1H NMR, 13C NMR, HRMS) analysis. These compounds were further biologically screened for their in vitro antibacterial activities against three pathogenic mycobacteria - M. abscessus S and R, M. marinum, and M. tuberculosis - as well as for their toxicity towards murine macrophages by the resazurin microtiter assay (REMA). Among the 45 derivatives, 17 compounds (3 hydroxamic acids, 9 hydrazides, and 5O-alkyl/O-acyl protected hydroxamic acids) were nontoxic against murine macrophages. When tested for their antibacterial activity, hydroxamic acid 9 h was found to be the most potent inhibitor against M. abscessus S and R only. Regarding hydrazide series, only 7h was active against M. abscessus R, M. marinum and M. tuberculosis; while the O-acyl protected hydroxamic acid derivatives 14d and 15d displayed promising antibacterial activity against both M. marinum and M. tuberculosis. Since such hydroxamic- and hydrazide-chelating groups have been reported to impair the activity of the peptide deformylase, in silico molecular docking studies in M. tuberculosis peptide deformylase enzyme active site were further performed with 7h in order to predict the possible interaction mode and binding energy of this molecule at the molecular level.

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling.

The identification and validation of a small molecule's targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule's targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ∼200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.

Intrabacterial lipid inclusions in mycobacteria: unexpected key players in survival and pathogenesis?

Mycobacterial species, including Mycobacterium tuberculosis, rely on lipids to survive and chronically persist within their hosts. Upon infection, opportunistic and strict pathogenic mycobacteria exploit metabolic pathways to import and process host-derived free fatty acids, subsequently stored as triacylglycerols in the form of intrabacterial lipid inclusions (ILI). Under nutrient-limiting conditions, ILI constitute a critical source of energy that fuels the carbon requirements and maintain redox homeostasis, promoting bacterial survival for extensive periods of time. In addition to their basic metabolic functions, these organelles display multiple other biological properties, emphasizing their central role in the mycobacterial life cycle. However, despite their importance, the dynamics of ILI metabolism and their contribution to mycobacterial adaptation/survival in the context of infection has not been thoroughly documented. Herein, we provide an overview of the historical ILI discoveries, their characterization and current knowledge regarding the microenvironmental stimuli conveying ILI formation, storage and degradation. We also review new biological systems to monitor the dynamics of ILI metabolism in extra- and intracellular mycobacteria and describe major molecular actors in triacylglycerol biosynthesis, maintenance and breakdown. Finally, emerging concepts regarding the role of ILI in mycobacterial survival, persistence, reactivation, antibiotic susceptibility and inter-individual transmission are also discussed.

Evaluation of plant growth promotion properties and induction of antioxidative defense mechanism by tea rhizobacteria of Darjeeling, India.

A total of 120 rhizobacteria were isolated from seven different tea estates of Darjeeling, West Bengal, India. Based on a functional screening of in vitro plant growth-promoting (PGP) activities, thirty potential rhizobacterial isolates were selected for in-planta evaluation of PGP activities in rice and maize crops. All the thirty rhizobacterial isolates were identified using partial 16S rRNA gene sequencing. Out of thirty rhizobacteria, sixteen (53.3%) isolates belong to genus Bacillus, five (16.6%) represent genus Staphylococcus, three (10%) represent genus Ochrobactrum, and one (3.3%) isolate each belongs to genera Pseudomonas, Lysinibacillus, Micrococcus, Leifsonia, Exiguobacterium, and Arthrobacter. Treatment of rice and maize seedlings with these thirty rhizobacterial isolates resulted in growth promotion. Besides, rhizobacterial treatment in rice triggered enzymatic [ascorbate peroxidase (APX), catalase (CAT), chitinase, and phenylalanine ammonia-lyase (PAL)], and non-enzymatic [proline and polyphenolics] antioxidative defense reactions indicating their possible role in the reduction of reactive oxygen species (ROS) burden and thereby priming of plants towards stress mitigation. To understand such a possibility, we tested the effect of rhizobacterial consortia on biotic stress tolerance of rice against necrotrophic fungi, Rhizoctonia solani AG1-IA. Our results indicated that the pretreatment with rhizobacterial consortia increased resistance of the rice plants towards the common foliar pathogen like R. solani AG1-IA. This study supports the idea of the application of plant growth-promoting rhizobacterial consortia in sustainable crop practice through the management of biotic stress under field conditions.

Dissecting the antibacterial activity of oxadiazolone-core derivatives against Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus), a rapidly growing mycobacterium, is an emergent opportunistic pathogen responsible for chronic bronchopulmonary infections in individuals with respiratory diseases such as cystic fibrosis. Most treatments of M. abscessus pulmonary infections are poorly effective due to the intrinsic resistance of this bacteria against a broad range of antibiotics including anti-tuberculosis agents. Consequently, the number of drugs that are efficient against M. abscessus remains limited. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs impair extracellular M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or act intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., compound iBpPPOX, via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis. Among them, the Ag85C protein has been confirmed as a vulnerable target of iBpPPOX. This study clearly emphasizes the potential of the OX derivatives to inhibit the extracellular and/or intracellular growth of M. abscessus by targeting various enzymes potentially involved in many physiological processes of this most drug-resistant mycobacterial species.

A simple method to produce Synechocystis PCC6803 biofilm under laboratory conditions for electron microscopic and functional studies.

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.

Molecular and culture-based surveys of metabolically active hydrocarbon-degrading archaeal communities in Sundarban mangrove sediments.

Archaea remain important players in global biogeochemical cycles worldwide, including in the highly productive mangrove estuarine ecosystems. In the present study, we have explored the diversity, distribution, and function of the metabolically active fraction of the resident archaeal community of the Sundarban mangrove ecosystem, using both culture-independent and culture-dependent approaches. To evaluate the diversity and distribution pattern of the active archaeal communities, RNA based analysis of the 16S rRNA gene was performed on an Illumina platform. The active Crenarchaeal community was observed to remain constant while active Euryarchaeal community underwent considerable change across the sampling sites depending on varying anthropogenic factors. Haloarchaea were the predominant group in hydrocarbon polluted sediments, leading us to successfully isolate eleven p-hydroxybenzoic acid degrading haloarchaeal species. The isolates could also survive in benzoic acid, naphthalene, and o-phthalate. Quantitative estimation of p-hydroxybenzoic acid degradation was studied on select isolates, and their ability to reduce COD of polluted saline waters of Sundarban was also evaluated. To our knowledge, this is the first ever study combining culture-independent (Next Generation sequencing and metatranscriptome) and culture-dependent analyses for an assessment of archaeal function in the sediment of Sundarban.

A single plasmid based CRISPR interference in Synechocystis 6803 - A proof of concept.

We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a "proof of concept". Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803.

Effective rhizoinoculation and biofilm formation by arsenic immobilizing halophilic plant growth promoting bacteria (PGPB) isolated from mangrove rhizosphere: A step towards arsenic rhizoremediation.

Arsenic (As) uptake by plants is largely influenced by the presence of microbial consortia and their interactions with As. In the coastal region of Bengal deltaic plain of Eastern India, the As-contaminated groundwater is frequently used for irrigation purposes resulting in an elevated level of soil As in agricultural lands. The health hazards associated with As necessitates development of cost-effective remediation strategies to reclaim contaminated agricultural lands. Among the available technologies developed in recent times, bioremediation using bacteria has been found to be the most propitious. In this study, two As-resistant halophilic bacterial strains Kocuria flava AB402 and Bacillus vietnamensis AB403 were isolated, identified and characterized from mangrove rhizosphere of Sundarban. The isolates, AB402 and AB403, could tolerate 35mM and 20mM of arsenite, respectively. The effect of As on the exopolysaccharide (EPS) synthesis, biofilm formation, and root association was evaluated for both the bacterial strains. Arsenic adsorption on the cell surfaces and intracellular accumulation in both the bacterial strains were promising under culture conditions. Moreover, both the strains when used as inoculum, not only promoted the growth of rice seedlings but also decreased As uptake and accumulation in plants.

Genome-Guided Insights into the Plant Growth Promotion Capabilities of the Physiologically Versatile Bacillus aryabhattai Strain AB211.

Bacillus aryabhattai AB211 is a plant growth promoting, Gram-positive firmicute, isolated from the rhizosphere of tea (Camellia sinensis), one of the oldest perennial crops and a major non-alcoholic beverage widely consumed all over the world. The whole genome of B. aryabhattai AB211 was sequenced, annotated and evaluated with special focus on genomic elements related to plant microbe interaction. It's genome sequence reveals the presence of a 5,403,026 bp chromosome. A total of 5226 putative protein-coding sequences, 16 rRNA, 120 tRNA, 8 ncRNAs, 58 non-protein coding genes, and 11 prophage regions were identified. Genome sequence comparisons between strain AB211 and other related environmental strains of B. aryabhattai, identified about 3558 genes conserved among all B. aryabhattai genomes analyzed. Most of the common genes involved in plant growth promotion activities were found to be present within core genes of all the genomes used for comparison, illustrating possible common plant growth promoting traits shared among all the strains of B. aryabhattai. Besides the core genes, some genes were exclusively identified in the genome of strain AB211. Functional annotation of the genes predicted in the strain AB211 revealed the presence of genes responsible for mineral phosphate solubilization, siderophores, acetoin, butanediol, exopolysaccharides, flagella biosynthesis, surface attachment/biofilm formation, and indole acetic acid production, most of which were experimentally verified in the present study. Genome analysis and experimental evidence suggested that AB211 has robust central carbohydrate metabolism implying that this bacterium can efficiently utilize the root exudates and other organic materials as an energy source. Genes for the production of peroxidases, catalases, and superoxide dismutases, that confer resistance to oxidative stresses in plants were identified in AB211 genome. Besides these, genes for heat shock tolerance, cold shock tolerance, glycine-betaine production, and antibiotic/heavy metal resistance that enable bacteria to survive biotic/abiotic stress were also identified. Based on the genome sequence information and experimental evidence as presented in this study, strain AB211 appears to be metabolically diverse and exhibits tremendous potential as a plant growth promoting bacterium.

Brevibacillus sp. KUMAs2, a bacterial isolate for possible bioremediation of arsenic in rhizosphere.

Arsenic (As) contamination of soil and water has been considered as a major global environmental issue during last few decades. Among the various methods so far reported for reclamation of As contaminated rhizosphere soil, bioremediation using bacteria has been found to be most promising. An As resistant bacterial isolate Brevibacillus sp. KUMAs2 was obtained from As contaminated soil of Nadia, West Bengal, India, which could resist As(V) and As(III) a maximum of 265mM and 17mM, respectively. The strain could remove ~40 percent As under aerobic culture conditions. As resistant property in KUMAs2 was found to be plasmid-borne, which carried both As oxidizing and reducing genes. The strain could promote chilli plant growth under As contaminated soil environment by decreasing As accumulation in plant upon successful colonization in the rhizosphere, which suggests the possibility of using this isolate for successful bioremediation of As in the crop field.

Cadmium toxicity in Escherichia coli: Cell morphology, Z-ring formation and intracellular oxidative balance.

This article deals with toxicological study of cadmium (Cd) as CdCl(2) on the growth and cell morphology of Escherichia coli K-12 MG1655. The minimum inhibitory concentration of Cd was 15µM. When cadmium was added at mid-log phase, growth was completely inhibited at 0.6mM and 50% of the bacterial growth retardation was found at 0.3mM concentration. At sublethal dose of Cd (0.2mM), majority of the cells showed filamentous form, suggested the possible effect of Cd on cell division. AFM study of bacterial cell morphology revealed severe surface damage of the treated cells in comparison to untreated cells. The expression of FtsZ decreased both at transcriptional and translational levels with the time of Cd exposure, thus cell division was affected and as a result cells took filamentous form. Due to Cd exposure, the nucleoid segregation remained unaffected, but improper Z-ring formation was observed. Activities of peroxidase and superoxide dismutase significantly decreased in treated cells with exposure time, which might elevate intracellular ROS level, as a consequence metabolic dysfunction and toxic effect were resulted.