Responses of Cajanus cajan and rhizospheric N-cycling communities to bioinoculants

Background and aims

Bioinoculants are commonly used for enhancing crop productivity but little information is available on their effect on key microbial communities such as those involved in the cycling of nitrogen, a major plant nutrient. Here we developed a formulation combining different bioinoculants (Bacillus megaterium, Pseudomonas fluorescens and Trichoderma harzianum) and examined their effects on both Cajanus cajan growth and N-cycling microorganisms.

Methods

Seven bioinoculant combinations were evaluated in pots under field conditions, and their effects on plant growth were measured using various biometric parameters. The abundances of the total bacterial and crenarchaeal communities along with those involved in N-cycling were monitored by qPCR at vegetative, pre-flowering, flowering and maturity stages of the crop.

Results

A significant increase in growth of C. cajan was observed when treated with mixture of three bioinoculants with dry biomass and grain yield increase by 330?% and 238?%, respectively. The combination of three bioinoculants also increased the abundance of nitrogen fixers and denitrifiers towards the flowering and maturity stages.

Conclusions

The consortium of three bioinoculants increased plant growth and grain yield of C. cajan. These bioinoculants also had a positive effect on the abundance of several N-cycling microbial communities stressing the importance of understanding non-target effects of bioinoculants together with their impact on plant growth.     

Members of the Arabidopsis FAE1-like 3-ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae

Several 3-keto-synthases have been studied, including the soluble fatty acid synthases, those involved in polyketide synthesis, and the FAE1-like 3-ketoacyl-CoA synthases. All of these condensing enzymes have a common ancestor and an enzymatic mechanism that involves a catalytic triad consisting of Cys, His, and His/Asn. In contrast to the FAE1-like family of enzymes that mediate plant microsomal fatty acid elongation, the condensation step of elongation in animals and in fungi appears to be mediated by the Elop homologs. Curiously these proteins bear no resemblance to the well characterized 3-keto-synthases. There are three ELO genes in yeast that encode the homologous Elo1p, Elo2p, and Elo3p proteins. Elo2p and Elo3p are required for synthesis of the very long-chain fatty acids, and mutants lacking both Elo2p and Elo3p are inviable confirming that the very long-chain fatty acids are essential for cellular functions. In this study we show that heterologous expression of several Arabidopsis FAE1-like genes rescues the lethality of an elo2Deltaelo3Delta yeast mutant. We further demonstrate that FAE1 acts in conjunction with the 3-keto and trans-2,3-enoyl reductases of the elongase system. These studies indicate that even though the plant-specific FAE1 family of condensing enzymes evolved independently of the Elop family of condensing enzymes, they utilize the same reductases and presumably dehydratase that the Elop proteins rely upon.     

Molecular characterization of beta-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

The beta-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reaction-restriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A beta-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a 'B-like' enzyme. This is the first study to have investigated the genetic heterogeneity of the beta-lactamase genes of Y. enterocolitica.     

Karyotypic diversity and evolution of seven mahseer species (Cyprinidae) from India

Mahseer is a group of fish species that are well known as food and game fishes. The taxonomy of the mahseer species is confusing owing to the morphological variations and habitat adaptation. Detailed karyomorphological investigations have been carried out in seven species of mahseer, using karyotyping, Ag-NOR and fluorescent staining techniques. The basic diploid chromosome number (2n), in all mahseer species, was observed to be 100; however, the karyotype formula varied among the species, which were recorded as: 20m + 14sm + 22st + 44t (fundamental arm number, FN = 134) in Tor khudree ; 22m + 24sm + 24st + 30t (FN = 146) in Tor mussullah; 12m + 22sm + 14st + 52t (FN = 134) in Tor putitora ; 20m + 24sm + 24st + 32t (FN = 144) in Tor tor ; 20m + 30sm + 24st + 26t (FN = 150) in Tor chelynoides; 20m + 20sm + 20st + 40t (FN = 140) in Tor progeneius; and 20m + 18sm + 14st + 48t (FN = 138) in Neolissochilus hexagonolepis . Silver staining of the chromosomes revealed the presence of multiple nucleolar organizer regions (NOR) in these mahseer species. The highest number of NORs was observed in T. tor (four pairs of chromosomes), whereas the other six species possessed Ag-NOR signals on only two pairs of chromosomes. Although chromomycin A₃ (CMA₃) staining induced bright fluorescence signals on same Ag-NORs sites, with CMA₃, one additional signal was observed on the p arm of subtelocentric chromosomes in T. tor , T. chelynoides , T. progeneius and N. hexagonolepis , which may indicate the presence of inactive NOR in these species. The information on cytogenetic profile of these mahseer species is discussed in the light of cytotaxonomic implications and understanding the karyoevolution of these fish species.     

Population distribution of 45S and 5S rDNA in golden mahseer, Tor putitora: population-specific FISH marker

Chromosomal locations of major 45S and minor 5S ribosomal DNAs (rDNAs) and organization of 5S rRNA genes were analysed in five different populations of golden mahseers (Tor putitora) using fluorescence in situ hybridization (FISH) and Southern blot hybridization. All five populations of T. putitora (2n = 100) showed a similar type of macro-karyotype composed of 12 metacentric, 22 submetacentric, 14 subtelocentric and 52 telocentric chromosomes. Analysis of active nucleolar organizer regions (NORs) by silver staining did not show any differences in number and chromosomal position in different populations. But FISH data showed significant difference between the populations, four of the five populations showed six 18S (three pairs) and two 5S (one pair) signals with positional polymorphism, while one population showed eight 18S and four 5S signals, respectively. Southern blot data confirms that 5S rDNA clusters present on two different chromosome pairs in Kosi river population contain non-transcribed spacers (NTS) of same length. In the present study, simultaneous localization of 45S and 5S rDNA by in situ hybridization helped us to develop the discrete population-specific markers in different geographically isolated populations of T. putitora.     

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.     

Identification of Host-Dependent Survival Factors for Intracellular Mycobacterium tuberculosis through an siRNA Screen

The stable infection of host macrophages by Mycobacterium tuberculosis (Mtb) involves, and depends on, the attenuation of the diverse microbicidal responses mounted by the host cell. This is primarily achieved through targeted perturbations of the host cellular signaling machinery. Therefore, in view of the dependency of the pathogen on host molecules for its intracellular survival, we wanted to test whether targeting such factors could provide an alternate route for the therapeutic management of tuberculosis. To first identify components of the host signaling machinery that regulate intracellular survival of Mtb, we performed an siRNA screen against all known kinases and phosphatases in murine macrophages infected with the virulent strain, H37Rv. Several validated targets could be identified by this method where silencing led either to a significant decrease, or enhancement in the intracellular mycobacterial load. To further resolve the functional relevance of these targets, we also screened against these identified targets in cells infected with different strains of multiple drug-resistant mycobacteria which differed in terms of their intracellular growth properties. The results obtained subsequently allowed us to filter the core set of host regulatory molecules that functioned independently of the phenotypic variations exhibited by the pathogen. Then, using a combination of both in vitro and in vivo experimentation, we could demonstrate that at least some of these host factors provide attractive targets for anti-TB drug development. These results provide a “proof-of-concept” demonstration that targeting host factors subverted by intracellular Mtb provides an attractive and feasible strategy for the development of anti-tuberculosis drugs. Importantly, our findings also emphasize the advantage of such an approach by establishing its equal applicability to infections with Mtb strains exhibiting a range of phenotypic diversifications, including multiple drug-resistance. Thus the host factors identified here may potentially be exploited for the development of anti-tuberculosis drugs.