Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Volker S. Brozel


Bacillus, co-culture, diazotrophy, interaction, Stretpomyces


Biological Nitrogen Fixation is the process of conversion of atmospheric dinitrogen into ammonia which is performed by symbiotic as well as free-living diazotrophic bacteria. A previous project to isolate free-living nitrogen fixing bacteria from a natural prairie yielded Streptomyces which grew on nitrogen free medium. Genome sequencing of three isolates revealed the presence of a second 16S rRNA gene sequence aligning with Bacillus, however repeated streaking only yielded Streptomyces colonies. The aim of this research was to characterize the interaction of three Streptomyces isolates that grow under nitrogen-free conditions, and appeared to be associated with Bacillus. The separation of Bacillus was achieved due to formation of heat resistant endospores, but isolation of Streptomyces into purity could not be achieved despite multiple approaches. Several separation techniques such as the size occlusion method, Chloramphenicol treatment of resistant Streptomyces, and growth of Streptomyces spores indicated that Bacillus is very closely and tightly associated with Streptomyces, that it seems to either have an endophytic relationship or stay as hitchhiker. The analysis of nitrogen fixing ability of Bacillus was done using acetylene reduction assay, 15N2 isotope incorporation assay, growth in nitrogen free soft agar, and amplification of nifH by PCR. The weakly positive acetylene reduction result suggested weak ability to fix nitrogen, however the genome did not contain any nif genes. We studied the growth of Bacillus and Streptomyces in co-culture in nitrogen-rich (R2A) and nitrogen-limiting (NFA) conditions. Microscopic investigation showed the presence of free rods on R2A after extended incubation, but absence of free rods on NFA, suggesting that Bacillus could be localized inside Streptomyces filaments, with some release upon ageing of the cultures. However, the quantification of 16S rRNA of both Bacillus and Streptomyces by qPCR indicated a constant ratio of Bacillus to Streptomyces, even in very young cultures. The genomes of Bacillus (321B and 40B) and Streptomyces (321I and 40I) were used to analyze the predicted metabolic pathways, with various pathways indicating species-species interaction. Degradation of aromatic compound pathways were more numerous in Streptomyces than Bacillus. Metabolic pathways such as RNA degradation, amino acids degradation were found to be more prominent in Bacillus. Streptomyces had a Vancomycin group antibiotics biosynthesis pathway, whereas a Vancomycin resistance mechanism was present in Bacillus. A four gene phylogenetic tree of Bacillus 321B and 40B showed that they are very closely related to each other and fall in the same cluster as B. subtilis. A five gene phylogenetic tree of Streptomyces 321 showed its closeness to S. phaeochromogenes, and 40 was closer to S. umbrinus. This study has opened areas for further investigation on the physical location of the interaction that helps to determine whether Bacillus occurs inside Streptomyces filaments as an endophyte or is only present as close association.

Library of Congress Subject Headings

Bacillus (Bacteria)
Nitrogen -- Fixation.



Number of Pages



South Dakota State University



Rights Statement

In Copyright