Genomics and bioinformatic

R.M. Berka; B.A. Nelson; E.J. Zaretsky; W.T. Yoder; M.W. Rey.

"Genomics of Fusarium venenatum: an alternative fungal host for making enzymes". 

In, Applied Mycology & Biotechnology, Vol. 4, Fungal Genomics (Arora, D.K. and Khachatourians, G.G., eds.) Elsevier Science, Amsterdam (2003)

Abstract
Fusarium venenatum A3/5 (formerly F. graminearum Schwabe A3/5) has been used since 1985 as the commercial source of Quorn™ mycoprotein, a processed form of fungal mycelia applied in several human food products to simulate chunks of chicken or beef.  Regulatory approval of the organism for human consumption made it an attractive candidate to consider as a host for the production of industrial and food grade enzymes. Systems for genetic manipulation and transformation of F. venenatum cells have been developed together with several strong promoters and selectable markers for the introduction and expression of heterologous genes.  Recent marketing of a heterologous xylanase and a fungal trypsin have provided a "proof of concept" for F. venenatum as a useful alternative to more traditional fungal hosts such as Aspergillus niger or A. oryzae.  However, compared to the latter organisms and well-studied model fungi such as Neurospora crassa and A. nidulans, information regarding the genomics of F. venenatum is inadequate.  This chapter provides one of the first overviews of F. venenatum genomic information based on a compilation of expressed sequence tags and chromosomal gene sequences to initiate momentum for more comprehensive genome sequencing efforts.

R.M. Berka; X. Cui; C.Yanofsky.

"Genome-wide transcriptional changes associated with genetic alterations and nutritional supplementation affecting tryptophan metabolism in Bacillus subtilis."

Proc. Nat. Acad. Sci. USA, 100, 5682-5687 (2003)

Abstract
DNA microarrays comprising  95% of the Bacillus subtilis annotated protein coding ORFs were deployed to generate a series of snapshots of genomewide transcriptional changes that occur when cells are grown under various conditions that are expected to increase or decrease transcription of the trp operon segment of the aromatic supraoperon. Comparisons of global expression patterns were made between cells grown in the presence of indole acrylic acid, a specific inhibitor of tRNATrp charging; cells deficient in expression of the mtrB gene, which encodes the tryptophan-activated negative regulatory protein, TRAP; WT cells grown in the presence or absence of two or three of the aromatic amino acids; and cells harboring a tryptophanyl tRNA synthetase mutation conferring temperature-sensitive tryptophan-dependent growth. Our findings validate expected responses of the tryptophan biosynthetic genes and presumed regulatory interrelationships between genes in the different aromatic amino acid pathways and the histidine biosynthetic pathway. Using a combination of supervised and unsupervised statistical methods we identified  100 genes whose expression profiles were closely correlated with those of the genes in the trp operon. This finding suggests that expression of these genes is influenced directly or indirectly by regulatory events that affect or are a consequence of altered tryptophan metabolism.

"Methods to reduce variability in DNA microarray experiments."

Genome Biology, 3, 0048.1-0048.16 (2002)

Abstract
Microarray data are subject to multiple sources of variation, of which biological sources are of interest whereas most others are only confounding. Recent work has identified systematic sources of variation that are intensity-dependent and non-linear in nature. Systematic sources of variation are not limited to the differing properties of the cyanine dyes Cy5 and Cy3 as observed in cDNA arrays, but are the general case for both oligonucleotide microarray (Affymetrix GeneChips) and cDNA microarray data. Current normalization techniques are most often linear and therefore not capable of fully correcting for these effects.  We present here a simple and robust non-linear method for normalization using array signal distribution analysis and cubic splines. These methods compared favorably to normalization using robust local-linear regression (lowess). The application of these methods to oligonucleotide arrays reduced the relative error between replicates by 5-10% compared with a standard global normalization method. Application to cDNA arrays showed improvements over the standard method and over Cy3-Cy5 normalization based on dye-swap replication. In addition, a set of known differentially regulated genes was ranked higher by the t-test. In either cDNA or Affymetrix technology, signal-dependent bias was more than ten times greater than the observed print-tip or spatial effects.  Intensity-dependent normalization is important for both high-density oligonucleotide array and cDNA array data. Both the regression and spline-based methods described here performed bett er than existing linear methods when assessed on the variability of replicate arrays. Dye-swap normalization was less effective at Cy3-Cy5 normalization than either regression or spline-based methods alone.

R.M. Berka; J. Hahn; I. Draskovic; M. Persuh; X. Cui; A. Sloma; W. Widner; D. Dubnau.

"Microarrray analysis of the Bacillus subtilis K-state: genome-wide expression changes induced by ComK." 

Mol. Microbiol., 43, 1331-1345 (2002)

Abstract
In Bacillus subtilis, the competence transcription factor ComK activates its own transcription as well as the transcription of genes that encode DNA transport proteins. ComK is expressed in about 10% of the cells in a culture grown to competence. Using DNA microarrays representing  95% of the protein-coding open reading frames in B. subtilis, we compared the expression profiles of wild-type and comK strains, as well as of a mecA mutant (which produces active ComK in all the cells of the population) and a comK mecA double mutant. In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated. The use of reporter fusions has confirmed these results for several genes. Many of the ComK-regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK-box promoter motifs. In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources. From this and previous work, we conclude that the ComK regulon defines a growth-arrested st ate, distinct from sporulation, of which competence for genetic transformation is but one notable feature. We suggest that this is a unique adaptation to stress and that it be termed the 'K-state'.

H. Jarmer; R. Berka; S. Knudsen; H.H. Saxild.

"Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions."

FEMS Microbiol. Lett., 206, 197-200 (2002)

Abstract
DNA microarrays were used to analyze the changes in gene expression in Bacillus subtilis strain 168 when nitrogen limiting (glutamate) and nitrogen excess (ammonium plus glutamate) growth conditions were compared. Among more than 100 genes that were significantly induced during nitrogen starvation we detected the comG, comF, comE, nin-nucA and comK transcription units together with recA. DNA was added to B. subtilis grown in minimal medium with glutamate as the sole nitrogen source and it was demonstrated that the cells were competent. Based on these observations we propose a simplification of previously designed one-step transformation procedures for B. subtilis strain 168.