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Jo Handelsman

Publications and source records attributed to Jo Handelsman.

33 records · Page 2Linked to original sources
PubMed2004

Genetics of zwittermicin a production by Bacillus cereus.

Zwittermicin A represents a new chemical class of antibiotic and has diverse biological activities, including suppression of oomycete diseases of plants and potentiation of the insecticidal activity of Bacillus thuringiensis. To identify genes involved in zwittermicin A production, we generated 4,800 transposon mutants of B. cereus UW101C and screened them for zwittermicin A accumulation. Nine mutants did not produce detectable zwittermicin A, and one mutant produced eightfold more than the parent strain. The DNA flanking the transposon insertions in six of the nine nonproducing mutants contains significant sequence similarity to genes involved in peptide and polyketide antibiotic biosynthesis. The mutant that overproduced zwittermicin A contained a transposon insertion immediately upstream from a gene that encodes a deduced protein that is a member of the MarR family of transcriptional regulators. Three genes identified by the mutant analysis mapped to a region that was previously shown to carry the zwittermicin A self-resistance gene, zmaR, and a biosynthetic gene (E. A. Stohl, J. L. Milner, and J. Handelsman, Gene 237:403-411, 1999). Further sequencing of this region revealed genes proposed to encode zwittermicin A precursor biosynthetic enzymes, in particular, those involved in the formation of the aminomalonyl- and hydroxymalonyl-acyl carrier protein intermediates. Additionally, nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) homologs are present, suggesting that zwittermicin A is synthesized by a mixed NRPS/PKS pathway.

Bacillus cereus↗
PubMed2004

Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods.

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the gamma-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the alpha-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

Animals↗
PubMed2004

Integration of microbial ecology and statistics: a test to compare gene libraries.

Libraries of 16S rRNA genes provide insight into the membership of microbial communities. Statistical methods help to determine whether differences in library composition are artifacts of sampling or are due to underlying differences in the communities from which they are derived. To contribute to a growing statistical framework for comparing 16S rRNA libraries, we present a computer program, integral -LIBSHUFF, which calculates the integral form of the Cramér-von Mises statistic. This implementation builds upon the LIBSHUFF program, which uses an approximation of the statistic and makes a number of modifications that improve precision and accuracy. Once integral -LIBSHUFF calculates the P values, when pairwise comparisons are tested at the 0.05 level, the probability of falsely identifying a significant P value is 0.098 for a study with two libraries, 0.265 for three libraries, and 0.460 for four libraries. The potential negative effects of making the multiple pairwise comparisons necessitate correcting for the increased likelihood that differences between treatments are due to chance and do not reflect biological differences. Using integral -LIBSHUFF, we found that previously published 16S rRNA gene libraries constructed from Scottish and Wisconsin soils contained different bacterial lineages. We also analyzed the published libraries constructed for the zebrafish gut microflora and found statistically significant changes in the community during development of the host. These analyses illustrate the power of integral -LIBSHUFF to detect differences between communities, providing the basis for ecological inference about the association of soil productivity or host gene expression and microbial community composition.

Aeromonas↗
PubMed2004

Metagenomics: application of genomics to uncultured microorganisms.

Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na(+)(Li(+))/H(+) antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

Biotechnology↗
PubMed2004

Status of the microbial census.

Over the past 20 years, more than 78,000 16S rRNA gene sequences have been deposited in GenBank and the Ribosomal Database Project, making the 16S rRNA gene the most widely studied gene for reconstructing bacterial phylogeny. While there is a general appreciation that these sequences are largely unique and derived from diverse species of bacteria, there has not been a quantitative attempt to describe the extent of sequencing efforts to date. We constructed rarefaction curves for each bacterial phylum and for the entire bacterial domain to assess the current state of sampling and the relative taxonomic richness of each phylum. This analysis quantifies the general sense among microbiologists that we are a long way from a complete census of the bacteria on Earth. Moreover, the analysis indicates that current sampling strategies might not be the most effective ones to describe novel diversity because there remain numerous phyla that are globally distributed yet poorly sampled. Based on the current level of sampling, it is not possible to estimate the total number of bacterial species on Earth, but the minimum species richness is 35,498. Considering previous global species richness estimates of 10(7) to 10(9), we are certain that this estimate will increase with additional sequencing efforts. The data support previous calls for extensive surveys of multiple chemically disparate environments and of specific phylogenetic groups to advance the census most rapidly.

Bacteria↗
PubMed2004

Metagenomics: genomic analysis of microbial communities.

Uncultured microorganisms comprise the majority of the planet's biological diversity. Microorganisms represent two of the three domains of life and contain vast diversity that is the product of an estimated 3.8 billion years of evolution. In many environments, as many as 99% of the microorganisms cannot be cultured by standard techniques, and the uncultured fraction includes diverse organisms that are only distantly related to the cultured ones. Therefore, culture-independent methods are essential to understand the genetic diversity, population structure, and ecological roles of the majority of microorganisms. Metagenomics, or the culture-independent genomic analysis of an assemblage of microorganisms, has potential to answer fundamental questions in microbial ecology. This review describes progress toward understanding the biology of uncultured Bacteria, Archaea, and viruses through metagenomic analyses.

Archaea↗
PubMed2003

Biotechnological prospects from metagenomics.

The recognition that most microorganisms in the environment cannot be cultured by standard methods stimulated the development of metagenomics, which is the genomic analysis of uncultured microorganisms. Two types of analysis have been used to obtain information from metagenomic libraries: a function-driven approach, in which metagenomic libraries are initially screened for an expressed trait, and a sequence-driven approach, in which libraries are initially screened for particular DNA sequences. New antibiotics and enzymes are among the early discoveries from metagenomics. Future refinement of methods that enrich for genes of particular function will accelerate the rate of discovery of useful molecules.

Bacterial Proteins↗
PubMed2003

Use of a promoter trap to identify Bacillus cereus genes regulated by tomato seed exudate and a rhizosphere resident, Pseudomonas aureofaciens.

The goal of this study was to identify genes in Bacillus cereus, a bacterium commonly associated with plant seeds and roots, that are affected by compounds originating from a host plant, tomato, or another rhizosphere resident, Pseudomonas aureofaciens. We constructed a B. cereus chromosomal DNA library in a promoter-trap plasmid, pAD123, which contains a promoterless version of the green fluorescent protein (GFP) gene, gfpmut3a. The library was screened by using fluorescence-activated cell sorting for clones showing a change in GFP expression in response to either tomato seed exudate or culture supernatant of P. aureofaciens strain 30-84. We identified two clones carrying genes that were induced by the presence of tomato seed exudate and nine clones carrying genes that were repressed by P. aureofaciens culture supernatant. A clone chosen for further study contained an open reading frame, designated lipA, that encodes a deduced protein with a lipoprotein signal peptide sequence similar to lipoproteins in B. subtilis. Expression of gusA under control of the lipA promoter increased twofold when cells were exposed to tomato seed exudate and in a concentration-dependent manner when exposed to a mixture of amino acids. When the wild type and a 10-fold excess of a lipA mutant were applied together to tomato seeds, 2 days after planting, the wild type displayed medium-dependent culturability, whereas the lipA mutant was unaffected. This study demonstrates the power of a promoter trap to identify genes in a gram-positive bacterium that are regulated by the biotic environment and resulted in the discovery of lipA, a plant-regulated gene in B. cereus.

Bacillus cereus↗
PubMed2003

A census of rRNA genes and linked genomic sequences within a soil metagenomic library.

We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, alpha-Proteobacteria, and CFB groups; greater representation among the beta- and gamma-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

Bacteria↗
PubMed2002

Toward an understanding of microbial communities through analysis of communication networks.

Bacteria receive signals from diverse members of their biotic environment. They sense their own species through the process of quorum sensing, which detects the density of bacterial cells and regulates functions such as bioluminescence, virulence, and competence. Bacteria also respond to the presence of other microorganisms and eukaryotic hosts. Most studies of microbial communication focus on signaling between the microbe and one other organism for empirical simplicity and because few experimental systems offer the opportunity to study communication among various types of organisms. But in the real biological world, microorganisms must carry on multiple molecular conversations simultaneously between diverse organisms, thereby constructing communication networks. We propose that biocontrol of plant disease, the process of suppressing disease through application of a microorganism, offers a model for the study of communication among multiple organisms. Successful biocontrol requires the sending and receiving of signals between the biocontrol agent and the pathogen, plant host, and microbial community surrounding the host. We are using Bacillus cereus, a biocontrol agent, and the organisms it must interact with, to dissect a communication network. This system offers an excellent starting point for study because its members are defined and well studied. An understanding of signaling in the B. cereus biocontrol system may provide a model for network communication among organisms that share a habitat and provide a new angle of analysis for understanding the interconnections that define communities.

Bacillus cereus↗
PubMed2002

Isolation of antibiotics turbomycin a and B from a metagenomic library of soil microbial DNA.

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.

Anti-Bacterial Agents↗
PubMed1998

Mutants of Rhizobium tropici strain CIAT899 that do not induce chlorosis in plants.

Type B strains of Rhizobium tropici induce severe foliar chlorosis when applied at planting to seeds of symbiotic host and non-host dicotyledonous plants. A Tn5-induced mutant, designated CT4812, or R. tropici strain CIAT899 that was unable to induce chlorosis was isolated. Cloning and sequencing of the DNA flanking the transposon in CT4812 revealed that the Tn5 insertion is located in a gene similar to glnD, which encodes uridylyltransferase/uridylyl-removing enzyme in enteric bacteria. Two marker-exchange mutants with insertions in glnD also failed to induce chlorosis in bean (Phaseolus vulgaris) plants. The 5'-most insertion in glnD (in mutant strain ME330) abolished the ability of R. tropici to utilize nitrate as a sole carbon source, whereas a mutation in glnD further downstream (in mutant strain ME245) did not have an obvious effect on nitrate utilization. A gene similar to the Salmonella typhimurium virulence gene mviN overlaps the 3' end of the R. tropici glnD homologue. A mutation in mviN had no effect on the ability of CIAT899 to induce chlorosis in bean plants. Therefore the glnD homologue, but not mviN, appears to be required for induction of chlorosis in plants by R. tropici strain CIAT899. A high nitrogen: carbon ratio in the rhizosphere of bean plants also prevented R. tropici from inducing chlorosis in bean plants. Mutations in either the glnD homologue or mviN had no significant effect on root nodule formation or acetylene reduction activity. A mutation in mviN eliminated motility in R. tropici. The sequence data, the inability of the glnD mutant to utilize nitrate, and the role of the R. tropici glnD gene in chlorosis induction in plants, a process that is nitrogen regulated, suggest that glnD plays a role in nitrogen sensing in R. tropici as its homologues do in other organisms.

Amino Acid Sequence↗