Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.

Burkholderia species are extremely multidrug resistant, environmental bacteria with extraordinary bioremediation and biocontrol properties. At the same time, these bacteria cause serious opportunistic infections in vulnerable patient populations while some species can potentially be used as bioweapons. The complete DNA sequence of more than 10 Burkholderia genomes provides an opportunity to apply functional genomics to a collection of widely adaptable environmental bacteria thriving in diverse niches and establishing both symbiotic and pathogenic associations with many different organisms. However, extreme multidrug resistance hampers genetic manipulations in Burkholderia. We have developed and evaluated a mutagenesis system based on the homing endonuclease I-SceI to construct targeted, non-polar unmarked gene deletions in Burkholderia. Using the cystic fibrosis pathogen Burkholderia cenocepacia K56-2 as a model strain, we demonstrate this system allows for clean deletions of one or more genes within an operon and also the introduction of multiple deletions in the same strain. We anticipate this tool will have widespread environmental and biomedical applications, facilitating functional genomic studies and construction of safe strains for bioremediation and biocontrol, as well as clinical applications such as live vaccines for Burkholderia and other Gram-negative bacterial species.

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An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ. The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene. When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIH site generates an unchanging 5' end for the lacZ mRNA. In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of ,l-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences. Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions. Both multicopy plasmid (pTL61T) and single-copy lambda (ATL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.Gene fusions have become important tools for the analysis of gene regulation in both procaryotic and eucaryotic systems. In general, the attachment of the regulatory sites of a given gene upstream of a reporter gene that encodes an easily assayed enzyme facilitates the analysis of expression of the affixed gene. The reporter gene may be used in either transcriptional (operon) fusions, where it retains its own translational start site but is dependent on the attached DNA for transcription, or in translational (protein) fusions, where both its transcription and translation are dependent on signals in the attached upstream DNA. The history and a wide variety of specific applications of gene fusions have been reviewed by Silhavy and Beckwith (45).Although a variety of reporter genes have been used in the construction of gene fusions, the most common is lacZ of Escherichia coli. The product, 3-galactosidase, is stable in many cellular backgrounds and can be precisely quantitated by a very simple and sensitive colorimetric assay. Moreover, the construction of fusions to lacZ extends all the experimental convenience of the lac genetic system to any given gene. That is, by using the available lac technology, one can readily screen and select for altered levels of f-galactosidase produced from the gene fusion. This greatly facilitates the regulatory analysis of the upstream attached gene.Numerous vector systems have been described for the construction of gene fusions to lacZ (45). Both multicopy (plasmid) and single-copy (bacteriophage) vectors have been used to construct gene fusions by either in vivo or in vitro manipulations. We previously described a vector system for the in vitro construction of transcriptional fusions to lacZ which had some advantages over the then currently available vectors (26). Both plasmid (pTL25) and phage (XTL25) versions were assembled, but we have used primarily the single-copy vector...

The activity of the sigma subunit of the RNA polymerase of Bacillus subtilis decreases markedly during the first 2 hr of sporulation. Moreover, sigma activity remains deficient throughout the Early during bacterial sporulation, RNA polymerase of Bacillus subtilis undergoes a change in template specificity.RNA polymerase purified from vegetative cells transcribes both phage ye DNA and poly(dA-dT) in vitro, while enzyme from sporulating B. subtilis actively transcribes only the synthetic template (1). Vegetative RNA polymerase holoenzyme consists of f' and # subunits of about 150,000 daltons, two a subunits of 42,000 daltons, and a sigma subunit of 55,000 daltons (2). Phosphocellulose chromatography dissociates the sigma factor, a subunit necessary for transcription of ye DNA, from the core of polymerase (#'fla2) that actively transcribes poly(dA-dT). On the other hand, RNA polymerase core enzyme purified from cells harvested during the sixth hour of sporulation is missing a # subunit and contains a polypeptide of 110,000 daltons (2). Moreover, this enzyme from sporulating cells neither binds vegetative sigma factor (3) nor responds to sigma in vitro (2). This finding suggested a possible mechanism for the change in template specificity of polymerase. Alteration of the core of RNA polymerase would prevent functioning of the sigma factor and thereby cause the loss of vegetative template specificity early during sporulation.To test this idea, we purified RNA polymerase at various stages of growth and sporulation. We report that the change in template specificity occurs during the first hour after the end 1865 of logarithmic growth, but that alteration of the core of polymerase does not commence until the second hour of sporulation. Brevet (in preparation) has independently made the same observation. Thus, the known alteration of core enzyme occurs too late to account for the loss of sigma activity. Furthermore, we find, by purifying RNA polymerase from a mixture of vegetative and sporulating bacteria separately labeled with two different radioisotopes, that the alteration of the # subunit may be accounted for by proteolytic cleavage in vitro during purification and that sporulating cells and spores contain polymerase with # subunits of molecular weight identical to that of vegetative polymerase. Possible mechanisms for the loss of sigma factor activity early during sporulation are discussed. METHODSMedia and Sporulation. Wild-type B. 8ubtilis strain NCTC 3610 (ATCC 6051), a Marburg strain, was used for ill the experiments. Vegetative and sporulating cells were grown in 121 B medium by the method of Sonenshein and Roscoe (4). For radioactive labeling of cells, the radioactive precursor was added during early logarithmic growth.The end of logarithmic growth is the start of the sporulation process and is defined as To. Refractile prespores began to appear after 5-6 hr, i.e., T5 and T6, and dormant spores were released at about T10-TI2. The total extent of sporulation after 24 hr was about 95%.Buffers were der...