Marcia S. Osburne

Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms

ABSTRACT Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium,Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

In Vitro Activity of Novel Rifamycins against Rifamycin-Resistant Staphylococcus aureus

ABSTRACT We describe novel rifamycin derivatives (new chemical entities [NCEs]) that retain significant activity against a comprehensive collection of Staphylococcus aureus strains that are resistant to rifamycins. This collection of resistant strains contains 21 of the 26 known single-amino-acid alterations in RpoB, the target of rifamycins. Some NCEs also demonstrated a lower frequency of resistance development than rifampin and rifalazil in S. aureus as measured in a resistance emergence test. When assayed for activity against the strongest rifamycin-resistant mutants, several NCEs had MICs of 2 μg/ml, in contrast to MICs of rifampin and rifalazil, which were 512 μg/ml for the same strains. The properties of these NCEs therefore demonstrate a significant improvement over those of earlier rifamycins, which have been limited primarily to combination therapy due to resistance development, and suggest a potential use of these NCEs for monotherapy in several clinical indications.

In Vitro Time-kill Activities of Rifalazil, Alone and in Combination with Vancomycin, against Logarithmic and Stationary Cultures of Staphylococcus aureus

Rifalazil is a novel rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the β subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that 1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, 2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log10 over either drug used alone and delayed the appearance of rifamycin-resistant mutants, 3) rifalazil and vancomycin in combination killed stationary phase cultures by 3∼4 Log10 by 48 hours.

Enhanced Activity of Rifalazil in Combination with Levofloxacin, Linezolid, or Mupirocin against Staphylococcus aureus In Vitro

Rifalazil is a potent second-generation ansamycin that kills bacterial cells by inhibiting the β subunit of RNA polymerase. Rifalazil has several improved properties compared with rifampicin, but retains rifampicins propensity to develop resistant mutants at high frequency. To explore strategies to overcome resistance development, we studied the effects of rifalazil in combination with several different antibiotics in an in vitro time-kill model, against both log phase and stationary phase Staphylococcus aureus cells. Experiments were carried out at high initial cell density so that the frequency and proliferation of resistant mutants could be monitored. We found that each combination was advantageous in terms of enhanced killing and the suppression of mutants, compared with each drug used alone. None of the three combinations was effective against stationary phase cells.

Preparation of fosmid libraries and functional metagenomic analysis of microbial community DNA.

One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries.

The Continuing Development of E. coli as a Heterologous Host for Complex Natural Product Biosynthesis

Heterologous biosynthesis of natural products is meant to enable access to the vast array of valuable properties associated with these compounds. Often motivated by limitations inherent in native production hosts, the heterologous biosynthetic process begins with a candidate host regarded as technically advanced relative to original producing organisms. Given this requirement, E. coli has been a top choice for heterologous biosynthesis attempts as associated recombinant tools emerged and continue to develop. However, success requires overcoming challenges associated with natural product formation, including complex biosynthetic pathways and the need for metabolic support. These two challenges have been heavily featured in cellular engineering efforts completed to position E. coli as a viable surrogate host. This chapter outlines steps taken to engineer E. coli with an emphasis on genetic manipulations designed to support the heterologous production of polyketide, nonribosomal peptide, and similarly complex natural products.

Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacterrodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not. Author summary Enterohemorrhagic Escherichia coli (EHEC), a food-borne pathogen that produces Shiga toxin, is associated with serious disease outbreaks worldwide, including over 390 food poisoning outbreaks in the U.S. in the last two decades. Humans acquire EHEC by ingesting contaminated food or water, or through contact with animals or their environment. Infection and toxin production may result in localized hemorrhagic colitis, but may progress to life-threatening systemic hemolytic uremic syndrome (HUS), the leading cause of kidney failure in children. Treatment for EHEC or HUS remains elusive, as antibiotics have been shown to exacerbate disease. Shiga toxin genes reside on a dormant bacterial virus present in the EHEC genome, but are expressed when the virus is induced to leave its dormant state and begin to replicate. Extensive virus replication has been thought necessary to produce sufficient toxin to cause disease. Using viral and bacterial mutants in our EHEC disease mouse model, we showed that whereas an inducing signal needed to begin viral replication was essential for lethal disease, virus production was not: sufficient Shiga toxin was produced to cause lethal mouse disease, even without viral replication. Future analyses of EHEC-infected human samples will determine whether this same phenomenon applies, potentially directing intervention strategies.

A mutation in the Bacillus subtilis rsbU gene that limits RNA synthesis during sporulation

Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacteriums ability to sporulate.IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.