Experimental genomics involves taking advantage of sequence information to investigate and understand the workings of genes, cells and organisms. We have developed an approach in which sequence information is used directly to design high-density, two-dimensional rays of synthetic oligonucleotides. The GeneChipe probe arrays are made using spatially patterned, light-directed combinatorial chemical synthesis and contain up to hundreds of thousands of different oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles. Here, we describe the fabrication of the arrays, their design and some specific applications to high-throughput genetic and cellular analysis.

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High density synthetic oligonucleotide arrays

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

http://science.sciencemag.org/content/sci/272/5265/1136.full.pdf

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

Structures and mechanisms of glycosyl hydrolases

cDNA microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment. DNA targets, in the form of 3' expressed sequence tags (ESTs), are arrayed onto glass slides (or membranes) and probed with fluorescent- or radioactively-labelled cDNAs. Here, we review technical aspects of cDNA microarrays, including the general principles, fabrication of the arrays, target labelling, image analysis and data extraction, management and mining.

Expression profiling using cdna microarrays

Bacterial biosorbents and biosorption

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended −10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide

Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.

Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

The bacterial envelope can be considered a combined mechanical and permeability barrier. This duality of function protects the cell against detrimental environmental influences and allows the maintenance of a high internal osmotic pressure. Although these properties imply rigidity, the barrier should also be flexible, permitting morphogenetic changes during growth and division and allowing for transport out of and into the bacterial cell. The mechanical integrity and permeability characteristics of the envelope are usually assigned separately to the two major constituents of the envelope: peptidoglycan is considered to be responsible for the mechanical integrity, whereas the membrane(s) constitute(s) the permeability barrier. Although this functional division is for the most part valid, it overlooks the possible contribution of peptidoglycan to the permeability characteristics of the envelope. For gram-positive bacteria, this issue was discussed in some early studies (30, 88), and the fact that the multilayered peptidoglycan, with its high degree of cross-linking and the presence of teichoic acids, might act as a permeability barrier is also the key feature of the recently proposed concept of gram-positive periplasm (68). For gram-negative bacteria, on the other hand, the permeability characteristics of peptidoglycan were overshadowed by those of the outer membrane (72). In fact, whereas low-molecular-weight compounds probably diffuse readily through peptidoglycan, the size of its pores may well be a limiting factor for the passage of larger proteins and protein complexes. In particular, the penetration of peptidoglycan by large multisubunit protein complexes that span the entire envelope may pose considerable steric problems. The presence of these envelopespanning structures has been demonstrated or postulated for a variety of transfer processes such as the secretion of toxins, the assembly of flagella and fimbriae, conjugation, and transformation. However, when the putative structure or function of these large multiprotein transfer machineries is described, the issue of peptidoglycan penetration is not usually taken into consideration. This is illustrated by the failure to depict the peptidoglycan layer in several schematic representations of these large putative envelope-spanning structures. The presence of peptidoglycan pores large enough to accommodate these structures is often taken for granted. On the other hand, some indirect evidence has led to speculation about possible peptidoglycan rearrangements during the assembly of these structures or during the actual transfer process (19, 26, 27, 36, 66). This minireview takes this speculation further by looking at the phenomenon of envelope spanning from a dimensional perspective: we focus on the correlation between the structural and functional features of peptidoglycan and the envelopespanning protein complexes. The observed dimensional discrepancies, combined with an examination of the reported links between the assembly of envelope-spanning complexes and peptidoglycan metabolism, strongly suggest the involvement of specific peptidoglycan-processing enzymes in this assembly. This hypothesis is supported by the recent identification of peptidoglycan hydrolase homologs in several crossenvelope transfer systems.

Wolfgang Keck

Papers

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays

Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide

Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

Peptidoglycan as a barrier to transenvelope transport.