Quantification of indole-3-acetic acid from plant associated <Emphasis Type="Italic">Bacillus</Emphasis> spp. and their phytostimulatory effect on <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Sixteen Bacillus strains isolated from rhizosphere, histoplane and phyllosphere of different plant species were identified by 16S rDNA gene sequencing and evaluated for in vitro auxin production as well as growth stimulation of Vigna radiata (L.) Wilczek. Auxin production by Bacillus spp. in L-broth medium supplemented with 1,000 μg ml⁻¹ L-tryptophan ranges from 0.60 to 3.0 μg IAA ml⁻¹ as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. Rhizospheric isolates exhibit relatively more IAA synthesis than histoplane and phyllosphere isolates. Plant microbe interaction experiments conducted under gnotobiotic conditions recorded 55.55, 46.46 and 46.20% increase in shoot length with Bacillus megaterium MiR-4, B. pumilus NpR-1 and B. subtilis TpP-1, respectively, over control. Bacillus inoculations also increased shoot fresh weight with B. megaterium MiR-4 (60.94%) and B. pumilus NpR-1 (37.76%). Highly significant positive correlation between auxin production analyzed by GC–MS and shoot length (r = 0.687**, P = 0.01) and shoot fresh weight (r = 0.703**, P = 0.01) was noted under gnotobiotic conditions. Similarly, significant correlation was also found between auxin production by Bacillus spp. (GC–MS analysis) and different growth parameters such as shoot length (r = 0.495*, P = 0.05), number of pods (r = 0.498*, P = 0.05) and grain weight (r = 0.537*, P = 0.05) at full maturity under natural wire house conditions. Results showed that auxin production potential of plant associated Bacillus spp. can be effectively exploited to enhance the growth and yield of V. radiata.     

The preponderance of P450s in the Mycobacterium tuberculosis genome

The genome of Mycobacterium tuberculosis (Mtb) encodes 20 different cytochrome P450 enzymes (P450s). P450s are mono-oxygenases, which are historically considered to facilitate prokaryotic usage of unusual carbon sources. However, their preponderance in Mtb strongly indicates crucial physiological functions, as does the fact that polycyclic azoles (known P450 inhibitors) have potent anti-mycobacterial effects. Recent structural and enzyme characterization data reveal novel features for at least two Mtb P450s (CYP121 and CYP51). Genome analysis, knockout studies and structural comparisons signify important roles in cell biology and pathogenesis for various P450s and redox partner enzymes in Mtb. Elucidation of structure, function and metabolic roles will be essential in targeting the P450s as an 'Achilles heel' in this major human pathogen.     

Deletion of cscR in Escherichia coli W improves growth and poly-3-hydroxybutyrate (PHB) production from sucrose in fed batch culture

Sucrose has several advantages over glucose as a feedstock for bioprocesses, both environmentally and economically. However, most industrial Escherichia coli strains are unable to utilize sucrose. E. coli W can grow on sucrose but stops growing when sucrose concentrations become low. This is undesirable in fed-batch conditions where sugar levels are low between feeding pulses. Sucrose uptake rates were improved by removal of the cscR gene, which encodes a protein that represses expression of the sucrose utilization genes at low sucrose concentrations. Poly-3-hydroxybutyrate (PHB) was used as a model compound in order to assess the effect of improved sugar utilization on bio-production. In the cscR knockout strain, production from sucrose was improved by 50%; this strain also produced 30% more PHB than the wild-type using glucose. This result demonstrates the feasibility of utilizing sucrose as an industrial feedstock for E. coli-based bioprocesses in high cell density culture.     

Deep Sequencing of HIV-1 near Full-Length Proviral Genomes Identifies High Rates of BF1 Recombinants Including Two Novel Circulating Recombinant Forms (CRF) 70_BF1 and a Disseminating 71_BF1 among Blood Donors in Pernambuco,Brazil

Background

The findings of frequent circulation of HIV-1 subclade F1 viruses and the scarcity of BF1 recombinant viruses based on pol subgenomic fragment sequencing among blood donors in Pernambuco (PE), Northeast of Brazil, were reported recently. Here, we aimed to determine whether the classification of these strains (n = 26) extends to the whole genome sequences.

Methods

Five overlapping amplicons spanning the HIV near full-length genomes (NFLGs) were PCR amplified from peripheral blood mononuclear cells (PBMCs) of 26 blood donors. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol. The prevalence of viral variants containing drug resistant mutations (DRMs) was compared between plasma and PBMCs.

Results

Of the 26 samples studied, 20 NFLGs and 4 partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Two distinct BF1 recombinant profiles designated CRF70_BF1 and CRF71_BF1, with 4 samples in profile I and 11 in profile II were detected and thus constitute two novel recombinant forms circulating in PE. Evidence of dual infections was detected in four patients co-infected with distinct HIV-1 subtypes. According to our estimate, the new CRF71_BF1 accounts for 10% of the HIV-1 circulating strains among blood donors in PE. Discordant data between the plasma and PBMCs-virus were found in 15 of 24 donors. Six of these strains displayed major DRMs only in PBMCs and four of which had detectable DRMs changes at prevalence between 1-20% of the sequenced population.

Conclusions

The high percentage of the new RF71_BF1 and other BF1 recombinants found among blood donors in Pernambuco, coupled with high rates of transmitted DRMs and dual infections confirm the need for effective surveillance to monitor the prevalence and distribution of HIV variants in a variety of settings in Brazil.     

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100^cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100^cue8 identified a second mutation within the same gene, tic100^soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100^soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100^cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Complementary mutations in TIC100 of the chloroplast inner envelope membrane cause reductions or corrective improvements in chloroplast protein import, and highlight a signaling role.

IN A NUTSHELLBackground: Plants harvest energy from the sun and CO₂ from the air and convert them into the energy-rich molecules they, and eventually us, are made of. Plants do this, photosynthesis, in bodies called chloroplasts inside their cells. Chloroplasts, made of protein and membrane material, were, before plants evolved, free-living bacteria, but the synthesis of most of their proteins occurs outside them, using information carried by the cell’s nuclear DNA, so most proteins have to be brought into developing chloroplasts, across the double membrane surrounding them, through dedicated, selective channels, formed by TOC (outer) and TIC (inner envelope) proteins. The identity of those channels matters as it helps determine versions of chloroplasts suited for particular environments. Which TIC proteins constitute the inner envelope channel has been a matter of controversy.Question: A mutant Arabidopsis plant called cue8 is slow-to-green (young leaves begin almost white) and shows delayed chloroplast and plant development. We looked for the molecular identity of the CUE8 gene. We also caused further mutations in this mutant and searched whether any corrected the defects in cue8.Findings: We found the mutated gene causing the cue8 defects is the TIC100 gene. This is one essential component of the “TIC 1-MDa complex,” one of the two versions of the TIC import complex under debate. That complex is made of several proteins, all present at reduced levels in cue8. In laboratory assays in which proteins are imported into isolated chloroplasts, cue8 performed worse than normal plants for a photosynthetic and a housekeeping chloroplast protein. A corrective, “suppressor” mutant was identified, and it carried a second mutation in TIC100, one physically complementary to the first one. Both the single and the double (suppressed) mutant still were slow-to-green, which evidences a signaling role for import defects to the nucleus, making photosynthetic genes active or not.Next steps: Surprisingly the grasses, including the cereals, have one core protein of the TIC 1 MDa complex but not the rest (including TIC100). We don’t know how their TIC channels operate. We also need to learn how the information on the defect in protein import, which occurs at the chloroplast envelope, is relayed to the cell’s nucleus (but we do have some clues).