Analysis of the Heat Shock Response of Neisseria meningitidis with cDNA- and Oligonucleotide-Based DNA Microarrays

Abstract

Oligonucleotide-and cDNA-based microarrays comprising a subset of Neisseria meningitidis genes were assessed for study of the meningococcal heat shock response and found to be highly suitable for transcriptional profiling of N. meningitidis. Employing oligonucleotide arrays encompassing the entire genome of N. meningitidis, we analyzed the meningococcal heat shock response on a global scale and identified 55 heat shockderegulated open reading frames (34 induced and 21 repressed).Sequencing of the genomes of Neisseria meningitidis serogroup A and serogroup B strains provided us with a tremendously broad range of information (15,23). The next step is the elucidation of gene expression patterns and gene product function on a genome-wide scale. DNA microarrays offer an ideal tool for high-throughput investigation of gene regulation on the transcriptional level (for review, see references 4, 13, and 17). The two most commonly employed DNA microarray platforms are oligonucleotide and cDNA arrays. Here we performed a comparative analysis of the suitability of both technology platforms for transcriptional profiling of N. meningitidis.The two main features of DNA microarray performance are sensitivity (signal intensity) and specificity (ratio of specific to nonspecific hybridization). Additional care must be taken to standardize experimental conditions and to avoid the detection of false-positive signals (12). In order to validate gene expression modulations of N. meningitidis observed using cDNAbased and oligonucleotide-based microarrays, we first performed parallel hybridizations of identical RNA samples to the same slide. Microarrays containing probes specific for 60 genes selected from the published genome sequence of N. meningitidis serogroup B strain MC58 (23) were produced (Table 1). For cDNA-arrays, internal fragments of each open reading frame (ORF) (300 to 560 bp) were PCR amplified. For oligonucleotide arrays, oligonucleotides (40-mers, three per gene) comprising gene-specific internal fragments (covering 5Ј, central, and 3Ј parts) were designed. All oligonucleotides (manufactured by MWG-Biotech AG, Ebersberg, Germany) carried a C6 amino linker modification at the 5Ј end for covalent attachment to the slide surface. Each probe was spotted 5 (oligonucleotides) or 10 times (PCR products) per array using the Affymetrix 417 Arrayer (MWG-Biotech AG). PCR products were spotted on CMT-GAPS-Coated Slides (Corning, Wiesbaden, Germany), oligonucleotides were spotted on Super Aldehyde Slides (TeleChem International, Sunnyvale, Calif.), and the slides were processed according to the manufacturers' instructions.Cultures of N. meningitidis strain MC58 (24) were grown to mid-logarithmic growth phase (optical density at 600 nm [OD 600 ] ϭ 0.5/5 ϫ 10 8 CFU/ml) at 37°C in supplemented proteose peptone medium and RNA isolated as previously described (5). The RNA was split into two aliquots, and onehalf was labeled with Cy3-dCTP, the other with Cy5-dCTP (Amersham Pharmacia, Freiburg, Germany) during a firststrand reverse transcri...

Transcriptome Analysis of Neisseria meningitidis during Infection

“…Biological experiments and subsequent hybridizations were repeated five times, and data of the independent experiments were combined. ORFs, minimally exhibiting a 1.6-fold deregulation on average and being deregulated more than 1.6-fold in at least three experiments, were defined as differentially transcribed; this cutoff is based on previous results which showed a 1.6-fold difference to be detected at a level of confidence of above 99.7% (13).…”

“…The adherent bacteria were subsequently dissociated from host cells by treatment with trypsin-EDTA and harvested by centrifugation. RNA was isolated from the neisserial pellet as described previously (13). Control bacteria were treated identically to bacteria from infections, i.e., all incubations, and wash and centrifugation steps were performed, except that no host cells were present during incubation in RPMI medium with FCS (HBMEC controls) or without FCS (HEp-2 controls).…”

“…For each of the 2,158 ORFs present in the published genome of N. meningitidis serogroup B strain MC58 (32), oligonucleotide-based microarrays were manufactured as previously described (13). Three oligonucleotides (40-mers) per gene comprising gene-specific internal fragments (covering 5Ј, central, and 3Ј parts) were designed.…”

Effect ofNeisseria meningitidisFur Mutations on Global Control of Gene Transcription

“…(B) The frpB promoter regions were amplified from each of the strains described above and sequenced, and the sequences and elements inferred from the experimentally mapped ϩ1 transcriptional start site in the present study are reported and reveal a phase-variable C tract between the Ϫ10 and Ϫ35 promoter elements in the strains. 20,25). In the present study, we used comparative transcriptome analysis of the Fur-null mutant to identify target genes affected directly and indirectly by this regulator, and as such we have begun to dissect the regulatory networks of the Fur protein and its role in global responses in N. meningitidis.…”

Global Gene Expression Analysis of the Heat Shock Response in the Phytopathogen Xylella fastidiosa

“…With the availability of whole genome sequences, the global response to temperature increase has been studied at the transcriptional level in diverse bacteria, from model organisms such as E. coli (45) and Bacillus subtilis (22), through the bioremediation-involved bacterium Shewanella oneidensis (16), to diverse mammalian pathogens such as Campylobacter jejuni (51), group A Streptococcus (50), Mycoplasma pneumoniae (59), Yersinia pestis (33), and Neisseria meningitidis (19). These global analyses revealed not only the induction of the classical Hsps but also changes in the expression levels of genes related to other cellular functions, such as flagellum biogenesis, transcriptional regulation, phage-related proteins, membrane composition, and pathogenesis, to cite a few.…”