Samina Mehnaz

MS/MS networking guided analysis of molecule and gene cluster families

Significance The paper introduces the concepts of molecular families (MFs) and gene cluster families (GCFs). We define MFs as structurally related molecules based on their mass spectral fragmentation patterns, whereas GCFs are biosynthetic gene clusters that show similar gene cluster organization with a high degree of sequence similarity. We use MS/MS networking as a tool to map the molecular network of more than 60 organisms, most of which are unsequenced, and locate their nonribosomal peptide MFs. These MFs from unsequenced organisms are then connected to GCFs of publicly available genome sequences of closely related organisms. The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779T. The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family–gene cluster families of hundreds or more diverse organisms in one single MS/MS network.

Association of nitrogen-fixing, plant-growth-promoting rhizobacteria (PGPR) with kallar grass and rice

Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using 15N isotopic dilution were carried out in nutrient solution and saline soil. These studies indicated an agronomically significant amount of nitrogen being fixed in soil. Kallar grass has a similar growth habitat to rice. Therefore similar studies were carried out with rice after isolating various diazotrophs from the roots which were also screened for their ability to produce auxin (IAA). Five such strains namely Azospirillum lipoferum N-4, Azospirillum brasilense Wb-3, Azoarcus K-1, Pseudomonas 96–51, Zoogloea Ky-1 were selected for inoculating two rice varieties i.e. NIAB-6 and BAS-370 under aseptic laboratory conditions. The nitrogen fixed was quantified using the 15N isotopic dilution method. Variety BAS-370 had nearly 70% nitrogen derived from atmosphere (Ndfa) when inoculated with Azospirillum N-4. Similar studies with the mixed inoculum using 15N fertilizer in the micro plots indicated that nearly 29% of plant nitrogen was derived from the atmosphere.

Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.

Lahorenoic acids A–C, ortho-dialkyl-substituted aromatic acids from the biocontrol strain Pseudomonas aurantiaca PB-St2

Three new aromatic acids, named lahorenoic acids A (1), B (2), and C (3), have been isolated along with the known compounds phenazine-1-carboxylic acid (4), 2-hydroxyphenazine-1-carboxylic acid (5), 2-hydroxyphenazine (6), 2,8-dihydroxyphenazine (7), cyclo-Pro-Tyr (8), cyclo-Pro-Val (9), cyclo-Pro-Met (10), and WLIP (11) and characterized from the biocontrol strain Pseudomonas aurantiaca PB-St2. The structures of these compounds were deduced by 1D and 2D NMR spectroscopic and mass spectral data interpretation. Compounds 2, 4, and 7 showed moderate antibacterial activity against mycobacteria and other Gram-positive bacteria, while 4 was also found to exhibit cytotoxic and antifungal properties.

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp.

Isolation and identification of diazotrophic bacteria from rice, wheat and kallar grass

Three bacterial isolates were obtained from the roots of rice, wheat and kallar grass. These isolates fix nitrogen in pure culture as confirmed by acetylene reduction assay in semi-solid nitrogen-free growth media. Production of growth hormones by these isolates was confirmed by colorimetric method. The isolates from wheat (strain Wb-3) and rice (strain N-4) were identified as Azospirillum brasilense and A. lipoferum, respectively, on the basis of their morphology, carbon utilization pattern and biochemical tests. The isolate K-1 from kallar grass, previously identified as Azospirillum brasilense, was confirmed to belong to a newly named genus Azoarcus (Reinhold-Hurek et al., 1993) by using 16S rRNA-targeted oligonucleotide probes and some morphological and physiological characteristics.

Genome sequence of the mycorrhizal helper bacterium Pseudomonas fluorescens BBc6R8

ABSTRACT We report the draft genome sequence of the mycorrhizal helper bacterium Pseudomonas fluorescens strain BBc6R8. This is the first genome of a mycorrhizal helper bacterium. The draft genome contains 6,952,353 bp and is predicted to encode 6,317 open reading frames. Comparative genomic analyses will help to identify helper traits.

Microbes – friends and foes of sugarcane

Sugarcane is an important cash crop for many countries because it is a major source of several products including sugar and bioethanol. To obtain maximum yields there is a need to apply large quantities of chemical fertilizers.Worldwide yields are also severely affected by more than sixty diseases, mostly caused by fungi but viruses, phytoplasmas, nematodes and other pests can also damage this crop. For most of these diseases, chemical control is not available and breeders are struggling with the development of pest resistant varieties. Many members of the grass family Poaceae establish associations with beneficial microbes which promote their growth by direct and indirect mechanisms. They can be used as means to reduce the need for chemical fertilizer and to minimize the impacts of pathogen invasion. This review highlights the diversity of the microbes associated with sugarcane and the role of beneficial microbes for growth promotion and biocontrol. More extensive use of beneficial microbes will help the sugarcane grower not only to reduce the use of chemical fertilizers but also minimize the disease. In this paper, a brief description of both the non‐pathogenic and pathogenic microbes associated with sugarcane is provided. Future prospects for the expanded use of beneficial microbes for sugarcane are also discussed and detailed herein.

The contribution of genome mining strategies to the understanding of active principles of PGPR strains

Pathogenic microorganisms and insects affecting plant health are a major and chronic threat to food production and the ecosystem worldwide. As agricultural production has intensified over the years, the use of agrochemicals has in turn increased. However, this extensive usage has had several detrimental effects, with a pervasive environmental impact and the emergence of pathogen resistance. In addition, there is an increasing tendency among consumers to give preference to pesticide-free food products. Biological control, through the employment of plant growth-promoting rhizobacteria (PGPR), is therefore considered a possible route to the reduction, even the elimination, of the use of agrochemicals. PGPR exert their beneficial influence by a multitude of mechanisms, often involving antibiotics and proteins, to defend the host plant against pathogens. To date, these key metabolites have been uncovered only by systematic investigation or by serendipity; their discovery has nevertheless been propelled by the genomic revolution of recent years, as increasing numbers of genomic studies have been integrated into this field, facilitating a holistic view of this topic and the rapid identification of ecologically important metabolites. This review surveys the highlights and advances of genome-driven compound and protein discovery in the field of bacterial PGPR strains, and aims to advocate for the benefits of this strategy.

Complete Genome Sequence of the Sugar Cane Endophyte Pseudomonas aurantiaca PB-St2, a Disease-Suppressive Bacterium with Antifungal Activity toward the Plant Pathogen Colletotrichum falcatum

ABSTRACT The endophytic bacterium Pseudomonas aurantiaca PB-St2 exhibits antifungal activity and represents a biocontrol agent to suppress red rot disease of sugar cane. Here, we report the completely sequenced 6.6-Mb genome of P. aurantiaca PB-St2. The sequence contains a repertoire of biosynthetic genes for secondary metabolites that putatively contribute to its antagonistic activity and its plant-microbe interactions.