Characterizing the type 6 secretion system (T6SS) of E. cloacae SBP-8 and its role in pathogenesis and bacterial competition.

Despite the relevance of E. cloacae as an opportunistic pathogen, very little is known about its pathogenicity mechanism and the factors influencing its virulence. The mechanism of E. cloacae pathogenicity appears to be complex and multifactorial, with the presence of different putative virulence factors whose role is still not clear in the development of the disease. In this study, we systematically investigated the role of T6SS (type six secretion system) of E. cloacae SBP-8, an environmental isolate, in eukaryotic and bacterial cell interaction. Analysis of the genome sequence of E. cloacae SBP-8 revealed the presence of sets of genes coding for the expression of one complete T6SS cluster, which is similar to T6SS-1 cluster of E. cloacae ATCC 13047 (clinical isolates). In addition, an Hcp effector protein was detected in the secretome, and this secretion depended on ClpV, an Atpase of T6SS, confirming that strain SBP-8 produces functional T6SS. Deletion of T6SS-associated gene clpV did not induce any significant change in the life span and rate of colonization in C. elegans. No major significant change was observed in the expression profiling of antimicrobial genes (clec-60, clec-85, clec-87 and lys-1) and toll-like receptor (toll-1) gene, involved in stimulating an immune response against the pathogen. No difference in the ability to invade and proliferate in intestinal cells and phagocytosis by macrophages was observed. In addition, we demonstrated that the ability of E. cloacae SBP-8 to out-compete Escherichia coli was reliant upon its T6SS in contact-dependent manner. Our results show that T6SS of the environmental isolates is required for interbacterial competition but not for invasion and proliferation inside host cells.

Characterization of functional amyloid curli in biofilm formation of an environmental isolate Enterobacter cloacae SBP-8.

The biofilm formation by bacteria is a complex process that is strongly mediated by various genetic and environmental factors. Biofilms contribute to disease infestation, especially in chronic infections. It is, therefore important to understand the factors affecting biofilm formation. This study reports the role of a functional amyloid curli in biofilm formation at various abiotic surfaces, including medical devices, by an environmental isolate of Enterobacter cloacae (SBP-8) which has been known for its pathogenic potential. A knockout mutant of csgA, the gene encoding the major structural unit of curli, was created to study the effect of curli on biofilm formation by E. cloacae SBP-8. Our findings confirm the production of curli at 25 °C and 37 °C in the wild-type strain. We further investigated the role of curli in the attachment of E. cloacae SBP-8 to glass, enteral feeding tube, and foley latex catheter. Contrary to the previous studies reporting the curli production below 30 °C in the majority of biofilm-forming bacterial species, we observed its production in E. cloacae SBP-8 at 37 °C. The formation of more intense biofilm in wild-type strain on various surfaces compared to curli-deficient strain (ΔcsgA) at both 25 °C and 37 °C suggested a prominent role of curli in biofilm formation. Further, electron and confocal microscopy studies demonstrated the formation of diffused monolayers of microbial cells on the abiotic surfaces by ΔcsgA strain as compared to the thick biofilm by respective wild-type strain, indicating the involvement of curli in biofilm formation by E. cloacae SBP-8. Overall, our findings provide insight into biofilm formation mediated by curli in E. cloacae SBP-8. Further, we show that it can be expressed at a physiological temperature on all surfaces, thereby indicating the potential role of curli in pathogenesis.

Elucidating the pathogenic potential of Enterobacter cloacae SBP-8 using Caenorhabditis elegans as a model host.

Enterobacter cloacae, an opportunistic nosocomial pathogen, is reported to possess different virulence factors that could potentially influence its pathogenesis. Generally, the E. cloacae infections are of endogenous origin occurring in immunocompromised patients. The mechanisms of pathogenicity remain elusive, possibly due to the absence of established model hosts. Thus, we explored the utility of Caenorhabditis elegans as a model host to test the pathogenicity of E. cloacae SBP-8, a soil isolate. E. cloacae SBP-8 progressively colonized the intestine of C. elegans. It induced cell death (as assessed through DNA damage), reproductive defect and reduction of lifespan, comparable to a clinical isolate, E. cloacae (MTCC 509). Observation with Nomarski microscopy revealed significant anterior pharyngeal distention, and altered egg arrangement with internal egg hatching in 70% infected worms. The internal egg hatching was observed as early as 48 h post infection. E. cloacae SBP-8 infection reduced the brood size by 16%. A 2',7'-dichlorodihydrofluorescein diacetate staining confirmed the 10-fold induction of reactive oxygen species implicating either mitochondrial damage or septic shock in infected worms. Expression analysis through RT-PCR indicated stimulation of immune response by E. cloacae SBP-8 in worms by upregulating tol-1, a Toll-like receptor, within 6 h of exposure. During the initial phase of infection (up to 24 h) the nematodes exhibited protective immune response by upregulating antimicrobial peptide genes, lys-1, clec-60, clec-85, and clec-87. However, these genes were downregulated at later hours (48 h), indicating the nematodes surrendered to the infection. A similar trend was observed for reproductive genes (lin-29 and let-23), suggesting a struggle to maintain functional reproduction by the nematodes. These results clearly demonstrate the pathogenic potential of E. cloacae SBP-8 and suggest the suitability of C. elegans as a model organism to study its pathogenesis. This is the first study indicating that E. cloacae infections could potentially originate from an exogenic source (here soil).

Biological evaluation and structure activity relationship of 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole derivatives as anti-leishmanial agents.

A series of piperazinyl-ß-carboline-3-carboxamide derivatives were designed through a molecular hybridization approach. Designed analogues were synthesized, characterized and evaluated for anti-leishmanial activity against Leishmania infantum and Leishmania donovani. In L. infantum inhibition assay, compounds 7d, 7g and 7c displayed potent inhibition of promastigotes (EC50 1.59, 1.47 and 3.73 µM respectively) and amastigotes (EC50 1.4, 1.9 and 2.6 µM respectively). SAR studies revealed that, para substitution of methoxy, chloro groups and methyl group on ortho position favored anti-leishmanial activity against L. infantum. Among these analogues 7d, 7h, 7n and 7g exhibited potent inhibition against L. donovani promastigotes (EC50 0.91, 4.0, 4.57 and 5.02 µM respectively), axenic amastigotes (EC50 0.9, 3.5, 2.2 and 3.8 µM respectively) and intracellular amastigotes (EC50 1.3, 7.8, 5.6 and 6.3 µM respectively). SAR studies suggested that, para substitution of methoxy group, para and meta substitution of chloro groups and benzyl replacement recommended for significant anti-leishmanial against L. donovani.

Synthesis and in-vitro anti-leishmanial activity of (4-arylpiperazin-1-yl)(1-(thiophen-2-yl)-9H-pyrido[3,4-b]indol-3-yl)methanone derivatives.

In the present study, we have reported synthesis and biological evaluation of a series of fifteen 1-(thiophen-2-yl)-9H-pyrido[3,4-b]indole derivatives against both promastigotes and amastigotes of Leishmania parasites responsible for visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis. Among these reported analogues, compounds 7b, 7c, 7f, 7g, 7i, 7j, 7m, 7o displayed potent activity (15.55, 7.70, 7.00, 3.80, 14.10, 9.25, 3.10, 4.85µM, respectively) against L. donovani promastigotes than standard drugs miltefosine (15.70µM) and pentamidine (32.70µM) with good selectivity index. In further, in-vitro evaluation against amastigote forms, two compounds 7g (8.80µM) and 7i (7.50µM) showed significant inhibition of L. donovani amastigotes. Standard drug amphotericin B is also used as control to compare inhibition potency of compounds against both promastigote (0.24µM) and amastigote (0.05µM) forms.

Sequential one-pot synthesis of bis(indolyl)glyoxylamides: Evaluation of antibacterial and anticancer activities.

A series of bis(indolyl)glyoxylamides 10a-n has been designed and synthesized. In situ generated indole-3-glyoxalylchloride from the reaction of readily available indole 9 with oxalyl chloride was treated with tryptamine to produce bis(indolyl)glyoxylamides 10a-n in 82-93% yields. All the synthesized bis(indolyl)glyoxylamides were well characterized and tested for their antibacterial activity against Gram-positive and Gram-negative bacterial strains. Compounds 10d, 10g and 10i were found to display potent antibacterial activity against Gram-negative strain. Further, the cytotoxicity of bis(indolyl)glyoxylamides 10a-n were evaluated against a panel of human cancer cell lines. Of the screened analogues, compound 10f (IC50=22.34µM; HeLa, 24.05µM; PC-3, 21.13µM; MDA-MB-231 and 29.94µM; BxPC-3) was identified as the most potent analogue of the series. Exposure of PC-3 cells to either 10a or 10f resulted in increased levels of cleaved PARP1, indicating that bis(indolyl)glyoxylamides induce apoptosis in PC-3 cells. Most importantly, compounds 10d, 10g and 10i were completely ineffective in mammalian cells, suggesting that they target bacterial-specific targets and thus will not display any toxicity in host cells.

Synthesis, evaluation and molecular docking studies of amino acid derived N-glycoconjugates as antibacterial agents.

Six amino acid derived N-glycoconjugates of d-glucose were synthesized, characterized and tested for antibacterial activity against G(+)ve (Bacillus cereus) as well as G(-)ve (Escherichia coli and Klebsiella pneumoniae) bacterial strains. All the tested compounds exhibited moderate to good antibacterial activity against these bacterial strains. The results were compared with the antibacterial activity of standard drug Chloramphenicol, where results of A5 (Tryptophan derived glycoconjugates) against E. coli and A4 (Isoleucine derived glycoconjugates) against K. pneumoniae bacterial strains are comparable with the standard drug molecule. In silico docking studies were also performed in order to understand the mode of action and binding interactions of these molecules. The docking studies revealed that, occupation of compound A5 at the ATP binding site of subunit GyrB (DNA gyrase, PDB ID: 3TTZ) via hydrophobic and hydrogen bonding interactions may be the reason for its significant in vitro antibacterial activity.

Secretory molecules from secretion systems fine-tune the host-beneficial bacteria (PGPRs) interaction.

Numerous bacterial species associate with plants through commensal, mutualistic, or parasitic association, affecting host physiology and health. The mechanism for such association is intricate and involves the secretion of multiple biochemical substances through dedicated protein systems called secretion systems SS. Eleven SS pathways deliver protein factors and enzymes in their immediate environment or host cells, as well as in competing microbial cells in a contact-dependent or independent fashion. These SS are instrumental in competition, initiation of infection, colonization, and establishment of association (positive or negative) with host organisms. The role of SS in infection and pathogenesis has been demonstrated for several phytopathogens, including Agrobacterium, Xanthomonas, Ralstonia, and Pseudomonas. Since there is overlap in mechanisms of establishing association with host plants, several studies have investigated the role of SSs in the interaction of plant and beneficial bacteria, including symbiotic rhizobia and plant growth bacteria (PGPB). Therefore, the present review updates the role of different SSs required for the colonization of beneficial bacteria such as rhizobia, Burkholderia, Pseudomonas, Herbaspirillum, etc., on or inside plants, which can lead to a long-term association. Most SS like T3SS, T4SS, T5SS, and T6SS are required for the antagonistic activity needed to prevent competing microbes, including phytopathogens, ameliorate biotic stress in plants, and produce substances for successful colonization. Others are required for chemotaxis, adherence, niche formation, and suppression of immune response to establish mutualistic association with host plants.

Biochemical and metabolic signatures are fundamental to drought adaptation in PGPR Enterobacter bugandensis WRS7.

Drought alone causes more annual loss in crop yield than the sum of all other environmental stresses. There is growing interest in harnessing the potential of stress-resilient PGPR in conferring plant resistance and enhancing crop productivity in drought-affected agroecosystems. A detailed understanding of the complex physiological and biochemical responses will open up the avenues to stress adaptation mechanisms of PGPR communities under drought. It will pave the way for rhizosphere engineering through metabolically engineered PGPR. Therefore, to reveal the physiological and metabolic networks in response to drought-mediated osmotic stress, we performed biochemical analyses and applied untargeted metabolomics to investigate the stress adaptation mechanisms of a PGPR Enterobacter bugendensis WRS7 (Eb WRS7). Drought caused oxidative stress and resulted in slower growth rates in Eb WRS7. However, Eb WRS7 could tolerate drought stress and did not show changes in cell morphology under stress conditions. Overproduction of ROS caused lipid peroxidation (increment in MDA) and eventually activated antioxidant systems and cell signalling cascades, which led to the accumulation of ions (Na+, K+, and Ca2+), osmolytes (proline, exopolysaccharides, betaine, and trehalose), and modulated lipid dynamics of the plasma membranes for osmosensing and osmoregulation, suggesting an osmotic stress adaption mechanism in PGPR Eb WRS7. Finally, GC-MS-based metabolite profiling and deregulated metabolic responses highlighted the role of osmolytes, ions, and intracellular metabolites in regulating Eb WRS7 metabolism. Our results suggest that understanding the role of metabolites and metabolic pathways can be exploited for future metabolic engineering of PGPR and developing bio inoculants for plant growth promotion under drought-affected agroecosystems.

Insights Into the Dynamics and Composition of Biofilm Formed by Environmental Isolate of Enterobacter cloacae.

Bacterial biofilms are clinically admissible and illustrate an influential role in infections, particularly those related to the implant of medical devices. The characterization of biofilms is important to understand the etiology of the diseases. Enterobacter cloacae are known for causing infections by forming biofilms on various abiotic surfaces, such as medical devices. However, a detailed characterization in terms of morphology and the molecular composition of the formed biofilms by this bacterium is sparse. The present study provides insights into the biofilm formation of E. cloacae SBP-8, an environmental isolate, on various surfaces. We performed assays to understand the biofilm-forming capability of the SBP-8 strain and characterized the adhering potential of the bacteria on the surface of different medical devices (foley latex catheter, enteral feeding tube, and glass) at different temperatures. We found that medical devices exhibited strong colonization by E. cloacae SBP-8. Using field emission-scanning electron microscopy (FE-SEM) studies, we characterized the biofilms as a function of time. It indicated stronger biofilm formation in terms of cellular density and EPS production on the surfaces. Further, we characterized the biofilm employing surface-enhanced Raman spectroscopy (SERS) and identified the vast heterogenic nature of the biofilm-forming molecules. Interestingly, we also found that this heterogeneity varies from the initial stages of biofilm formation until the maturation and dispersion. Our studies provide insights into biofilm composition over a period of time, which might aid in understanding the biofilm dispersion phases, to enhance the presently available treatment strategies.

ACC deaminase producing rhizobacterium Enterobacter cloacae ZNP-4 enhance abiotic stress tolerance in wheat plant.

Plant growth promoting rhizobacterium (PGPR) designated as ZNP-4, isolated from the rhizosphere of Ziziphus nummularia, was identified as Enterobacter cloacae following 16S rRNA sequence analysis. The isolated strain exhibited various plant growth promoting (PGP) traits. The 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activity was evaluated under diverse physiological conditions that could be useful for minimizing the abiotic stress-induced inhibitory effects on wheat plants. The strain showed resistance to salt (NaCl) and metal (ZnSO4) stress. The effect of E. cloacae ZNP-4 on the augmentation of plant growth was studied under salinity stress of 150 mM (T1 treatment) & 200 mM (T2 treatment) NaCl. The inoculation of strain ZNP-4 significantly improved the various growth parameters of wheat plant such as shoot length (41%), root length (31%), fresh weight (28%), dry weight (29%), photosynthetic pigments chlorophyll a (62%) and chlorophyll b (34%). Additionally, the strain was found to be efficient for minimizing the imposed Zn stress in terms of improving plant growth, biomass and photosynthetic pigments in pots containing different levels of metal stress of 150 mg kg-1 (treatment T1) and 250 mg kg-1 (treatment T2). Isolate ZNP-4 also improved the proline content and decreased malondialdehyde (MDA) level under both salinity and metal stress, therefore maintaining the membrane integrity. Furthermore, bacterial inoculation increased the activities of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). The positive effects of PGPR occurred concurrently with the decrease in abiotic stress-induced reactive oxygen species (ROS) molecules such as hydrogen peroxide (H2O2) and superoxide (O2-) contents. Overall, the observed results indicate that use of bacteria with such beneficial traits could be used as bio-fertilizers for many crops growing under stress conditions.

QbD-driven formulation development and evaluation of topical hydrogel containing ketoconazole loaded cubosomes.

The proposed study aimed to develop topical hydrogel containing ketoconazole loaded cubosomes with lower surfactant concentrations using the 'Quality by Design' (QbD) approach. Risk assessment was performed, followed by screening and optimization of formulations by 32 factorial design using Design-Expert® software. Keeping the combination of constituents similar to that of the optimized batches as predicted post conduct of 'Design of Experiment' (DoE) studies, scale-up batches were prepared. The 32 factorial design model successfully predicted the composition of the optimized formulation within the confidence limits. In vitro drug release study was performed and analyzed by various mathematical models. Ex vivo permeation study was investigated using goat ear skin. These ketoconazole loaded cubosomes showed a release pattern similar to the Korsmeyer-Peppas model experiencing Fickian diffusion having 67% cumulative ketoconazole release within 24 h. Ex vivo permeation study of hydrogel containing ketoconazole loaded cubosomes revealed a sustained release pattern through the goat ear skin with around 92.73 % release within 24 h. Scale-up studies also gave the confirmatory results for the post characterization studies, whereby the particle size of ketoconazole loaded cubosomes was 198 nm with 45% ketoconazole entrapment efficiency. This hydrogel containing ketoconazole loaded cubosomes can be used for topical drug delivery.

Priming with ACC-utilizing bacterium attenuated copper toxicity, improved oxidative stress tolerance, and increased phytoextraction capacity in wheat.

The major challenges for the plants growing in metal-contaminated soils are deficiency of nutrients, biomass reduction, and severe oxidative damages in the presence of heavy metals. In this regard, our aim was to overcome these challenges through the use of efficient microbial strains in metal-polluted soils and to assess its/their physiological and biochemical effects. In the current study, a copper (Cu)-resistant bacterium was isolated from the rhizospheric soil of 'Ziziphus nummularia' and evaluated for its ability to promote the wheat growth under the gradient stress of copper. Based on 16S rRNA gene sequencing, the isolate was identified as Pantoea sp. Among the plant growth promoting tests, the isolate showed the production of indole acetic acid, solubilization of inorganic phosphate, and ACC deaminase activity. Also, the isolate showed resistance to many heavy metals and antibiotics and increased the water-soluble copper in solution. The results of pot studies showed that bacterial application promoted various growth parameters of wheat plants and also enhanced the Cu uptake of wheat from the Cu-amended soil. The results showed that enhancement of Cu stress (100 to 300 mg kg-1) resulted in a decrease in various compatible solutes such as proline, total soluble sugars, and total protein content, and increase in the level of malondialdehyde (MDA), latter of which is the indicator of oxidative stress. Bacterial treatment markedly increased the proline, soluble sugar, total protein content, and decreased the MDA content under Cu stress. In addition, bacterial inoculation significantly alleviated the harmful effect of metal toxicity by decreasing the activation of ROS molecules including superoxide (O2-) and hydrogen peroxide (H2O2). The activation of various antioxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) was noted following bacterial inoculation under Cu stress. Therefore, the present study demonstrates the potential of the isolate Pantoea sp. ZNP-5 to improve the growth and phytoextraction of metal from the metal-polluted soil through the polyphasic mechanism of action.

The draft genome sequence of the plant growth promoting rhizospheric bacterium Enterobacter cloacae SBP-8.

Here, we report the draft genome sequence and annotation of plant growth promoting rhizobacterium Enterobacter cloacae SBP-8 isolated from the rhizosphere of Sorghum bicolor L. growing in desert region of Rajasthan, India. From the genome sequences, we identified the genes encoding plant growth promoting properties such as 1-aminocyclopropane-1-carboxylate deaminase (AcdS), phosphate solubilisation, siderophore, and IAA (indole acetic acid) production. The genes encoding different functions required for colonization including motility, chemotaxis, adherence, and secretion system (I, II, IV, VI) were also identified. The complete genome sequence of this bacterium consists of one chromosome (48,54,065 bp) and one plasmid (85,398). The genome sequence of Enterobacter cloacae SBP-8 was deposited in the Genbank with the accession number CP016906 (chromosome) and CP017413 (plasmid).

Quantitative proteomics analysis reveals the tolerance of wheat to salt stress in response to Enterobacter cloacae SBP-8.

Salinity stress adversely affects the plant growth and is a major constraint to agriculture. In the present study, we studied the role of plant growth promoting rhizobacterium (PGPR) Enterobacter cloacae SBP-8 possessing ACC deaminase activity on proteome profile of wheat (Triticum aestivum L.) under high salinity (200 mM NaCl) stress. The aim of study was to investigate the differential expressed protein in selected three (T-1, T-2, T-3) treatments and absolute quantification (MS/MS analysis) was used to detect statistically significant expressed proteins. In this study, we investigated the adaptation mechanisms of wheat seedlings exposed to high concentration of NaCl treatment (200 mM) for 15 days in response to bacterial inoculation based on proteomic data. The identified proteins were distributed in different cellular, biological and molecular functions. Under salt stress, proteins related to ion-transport, metabolic pathway, protein synthesis and defense responsive were increased to a certain extent. A broader comparison of the proteome of wheat plant under different treatments revealed that changes in some of the metabolic pathway may be involved in stress adaption in response to PGPR inoculation. Hierarchical cluster analysis identified the various up-regulated/down-regulated proteins into tested three treatments. Our results suggest that bacterial inoculation enhanced the ability of wheat plant to combat salt stress via regulation of transcription factors, promoting antioxidative activity, induction of defense enzymes, lignin biosynthesis, and acceleration of protein synthesis.

The Multifarious PGPR Serratia marcescens CDP-13 Augments Induced Systemic Resistance and Enhanced Salinity Tolerance of Wheat (Triticum aestivum L.).

The present study demonstrates the plant growth promoting (PGP) potential of a bacterial isolate CDP-13 isolated from 'Capparis decidua' plant, and its ability to protect plants from the deleterious effect of biotic and abiotic stressors. Based on 16S rRNA gene sequence analysis, the isolate was identified as Serratia marcescens. Among the PGP traits, the isolate was found to be positive for ACC deaminase activity, phosphate solubilization, production of siderophore, indole acetic acid production, nitrogen fixation, and ammonia production. CDP-13 showed growth at an increased salt (NaCl) concentration of up to 6%, indicating its potential to survive and associate with plants growing in saline soil. The inoculation of S. marcescens enhanced the growth of wheat plant under salinity stress (150-200 mM). It significantly reduced inhibition of plant growth (15 to 85%) caused by salt stressors. Application of CDP-13 also modulated concentration (20 to 75%) of different osmoprotectants (proline, malondialdehyde, total soluble sugar, total protein content, and indole acetic acid) in plants suggesting its role in enabling plants to tolerate salt stressors. In addition, bacterial inoculation also reduced the disease severity caused by fungal infection, which illustrated its ability to confer induced systemic resistance (ISR) in host plants. Treatment of wheat plants with the test organism caused alteration in anti-oxidative enzymes activities (Superoxide dismutase, Catalase, and Peroxidase) under various salinity levels, and therefore minimizes the salinity-induced oxidative damages to the plants. Colonization efficiency of strain CDP-13 was confirmed by CFU count, epi-fluorescence microscopy, and ERIC-PCR-based DNA fingerprinting approach. Hence, the study indicates that bacterium CDP-13 enhances plant growth, and has potential for the amelioration of salinity stress in wheat plants. Likewise, the results also provide insights into biotechnological approaches to using PGPR as an alternative to chemicals and pesticides.

The plant-growth-promoting bacterium Klebsiella sp. SBP-8 confers induced systemic tolerance in wheat (Triticum aestivum) under salt stress.

Plant-growth-promoting bacteria (PGPB) with 1-aminocyclopropane-1-carboxylatedeaminase (ACCD) activity can protect plants from the deleterious effects of abioticstressors. An ACCD bacterial strain, SBP-8, identified as Klebsiella sp., also having other plant-growth-promoting activities, was isolated from Sorghum bicolor growing in the desertregion of Rajasthan, India. ACCD activity of SBP-8 was characterized at biochemical, physiological, and molecular levels. The presence of AcdS, a structural gene for ACCD, was confirmed by the polymerase chain reaction. Strain SBP-8 showed optimum growth and ACCD activity at increased salt (NaCl) concentrations of up to 6%, indicating its potential to survive and associate with plants growing in saline soil. Inoculation of wheat plants with SBP-8 when grow in the presence of salt (150-200 mM) and temperature (30-40 °C) stressors resulted inamelioration of stress conditions by increasing plant biomass and chlorophyll content, and are duction in plant growth inhibition (10-100%) occurred due to salt and temperature stressors. Moreover, strain SBP-8 also caused Na(+) exclusion (65%) and increased uptake of K(+) (84.21%) in the host plant. This property can protect plants from adverse effects of Na(+) on plant growth and physiology. Thus, SBP-8 improves growth of the host plant and protects from salt stressors through more than one mechanism including an effect of ACCD activity and on K(+)/Na(+) ratio in plants. The colonization efficiency of strain SBP-8 was confirmedby CFU (colony-forming unit) count, microscopy, and ERIC-PCR based DNA-finger-printing approach. Therefore, and the use of efficient colonizing plant-growth-promoting bacteria may provideinsights into possible biotechnological approaches to decrease the impact of salinity and other stressors.

Inactivation of cyanobacterial nitrogenase after exposure to ultraviolet-B radiation.

Exposure of the N(2)-fixing cyanobacterium Anabaena BT2 to ultraviolet-B radiation (2.5 W m(-2)) for 30 min resulted in complete loss of nitrogenase activity but 100% cell killing occurred only after a 90-min exposure. Inactivation of nitrogenase activity was not specific to Anabaena BT2; other species also showed a similar effect. The time required for 100% killing and inactivation of nitrogenase activity differed in various species, and this difference may be ascribed to the presence of different levels of UV-B protection mechanisms in individual species. Inhibition of nitrogenase activity was immediate, since exposure of cultures to UV-B for as little as 5 min elicited some inhibition of activity. The activity of UV-B-inhibited nitrogenase did not recover upon transfer of exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment of inhibitors of protein synthesis and PS-II activity, it was demonstrated that restoration of nitrogenase activity in a UV-B-treated culture occurred by fresh synthesis of nitrogenase polypeptide. Our findings suggest that estimation of nitrogenase activity in diazotrophic species may be used as a marker enzyme for assessing the impact of UV-B radiation.