The current investigation was performed to identify the genes preferentially transcribed under FeHm limitation in two clinically relevant isolates of H. influenzae and, by comparison with previously published observations, to begin to define the core FeHm modulon for this species. Since our focus at the outset was the elucidation of potential targets mediating iron and/or heme uptake, our experimental conditions were optimized by Q-PCR analysis of known FeHm repressible genes. By inference, these conditions were expected to identify other genes with similar transcriptional kinetics.
This study identified a putative core set of 37 FeHm-ve genes that are similarly regulated in three unrelated H. influenzae isolates. This core contains the majority of the known FeHm uptake genes, including the hgps, the hxuCBA operon, and the tbp operon. All of these are TonB-dependent receptor complexes and the tonB operon was also determined to be FeHm-ve in all isolates examined.
Using the criteria of genes determined to be FeHm-ve with similarity to known TonB-dependent proteins two other genes were identified with a potential role in FeHm uptake; HI0113 and HI369. Both of these genes are subjects of ongoing studies in our laboratory
During the design of the experiments detailed in this manuscript, as well as those previously reported from strain Rd KW20 , great care was taken to ensure that each isolate was treated identically to minimize environmental influences other than the FeHm status of the media. Nevertheless, there remain potential strain-specific transcriptional differences observed among the three isolates by microarray. While every effort was taken to ensure that the observed effects result solely from the transition of the culture from FeHm restricted to FeHm supplemented conditions, other environmental stimuli cannot be entirely ruled out. It is possible that in our microarray experiments the removal of a large sample from the culture might perturb oxygen levels in the remaining medium. However, identical control experiments in which smaller samples were removed for Q-PCR analysis (0.5 ml, representing 0.4% culture volume) showed similar regulatory profiles of 94% of the core FeHm genes predicted by microarray. This finding indicates that oxygenation effects due to removal of a large sample volume in the microarray experiments are unlikely to account for the apparent effects of FeHm addition on gene regulation. A second potential environmental condition that could lead to transcriptional changes distinct from the response to FeHm addition is an alteration in the levels of other nutrients between the two time points. The two samples used in the microarray are separated by only a 20 minute period and are drawn from the same culture. This removes any potential flask-to-flask differences. In addition, due to the FeHm restriction, the cultures are growing slowly and are at a relatively low cell density and thus unlikely to be significantly depleting available nutrients during the time period between sampling. A further source of experimentally induced variation may be the temporal separation between individual microarray experiments. To overcome this, the control Q-PCR experiments were performed at the same time, with the same batch of media, under identical conditions. Taken together, these considerations suggest that nutrient depletion is unlikely to play a significant role in the observed outcomes of gene expression analyses. While we propose that the 20 minute period between sampling is too short to observe nutrient related changes in gene transcription in the culture, we have not performed experiments to specifically address this issue and are unable to completely discount this possibility.
Additional File 1 only contains genes for which a fold change >1.5 was determined by microarray in at least one isolate. However, the level of expression of the "unregulated", constitutively expressed FeHm genes (not shown in Additional File 1) is unclear. Are these genes expressed at a low constitutive level or at a level more like upregulated genes? In addition, the independent Q-PCR analysis also emphasize the fact that the reporting of "ns" in Additional File 1 for fold change of a gene is not an indication that that gene is not regulated. Thus, further studies are required to define the actual regulatory status of "ns" genes before the gene may be excluded as part of the core FeHm modulon. These missing pieces of information would have a profound effect on our understanding of the system biology of FeHm uptake and utilization as well as cellular metabolism.
Comparisons of the FeHm responsive genes in the three isolates indicate that NTHi R2866 and Hib10810 are more similar to each other than they are to Rd KW20. This may arise from the fact that the two isolates examined in this study are both recently isolated clinical isolates as opposed to Rd KW20 which has undergone multiple passages on artificial media or it may merely reflect the fact that Rd KW20 has a different mode of regulation than the other isolates. To investigate this aspect further, additional microarray analyses are planned with other genome-sequenced H. influenzae isolates.
In constrast with other model organisms, relatively few studies have been published in H. influenzae that have examined global transcriptional regulatory networks. Comparative genomic analyses have identified putative members of the FNR and CRP regulons  and purine, arginine and aromatic amino acid regulons . Knockouts have identified genes whose transcriptional profiles differ upon disruption of a possible regulatory element or co-effector. Included in these studies are the results of disruption of the tfoX (sxy) gene involved in regulating competence genes and the cya gene responsible for production of cAMP . This latter effect would alter regulation patterns of genes within the CRP regulon. Other studies have examined effects of disruption of the arcA and oxyR regulators [32, 33]. Furthermore, transcriptional effects resulting from specific environmental changes have been examined independently  or can be derived from the above studies. Thus, we can determine transcription patterns affected by transition between FeHm availability , sugar and nucleotide availability , and presence of oxidizing agents .
As could be expected, the results of our examination of transcriptional patterns altered by FeHm availability possess degrees of overlap with the results from these previous studies. For example, FeHm restriction can be predicted to result in a profound impact on energy generation in the cell. In fact, many genes previously demonstrated to be upregulated during nutrient limitation in a cAMP-dependent manner are also downregulated in this study upon the addition of FeHm. This suggests that the FeHm depleted cultures had elevated cAMP levels. Yet some genes demonstrate the opposite effect. These data show that gene regulatory networks in H. influenzae exhibit complexity beyond mere concurrent activation of independent regulons. For example, the dps gene (HI1427), encoding the DPS ferritin protein, appears to be part of the ArcA and OxyR regulons as well as responsive to FeHm levels. Transcripts from the operon comprising the ornithine decarboxylase and putrescine transporter (HI0591 and HI0592) and the HI0608 gene encoding a probable transport permease are negatively impacted by disruption of the arcA and cya genes compared to the wildtype strain and are also downregulated in response to FeHm addition; yet transcripts the gene encoding lactate permease (HI1218) which decrease in the cya mutant, increase in the arcA mutant and are upregulated in response to FeHm addition. Studies examining targets of particular transcriptional regulators should be performed in parallel with studies examining the effect of a particular stimulating substance(s) as part of a global approach to define gene regulation. However, the lack of sufficient data from H. influenzae makes a thorough examination of iron or heme's affect on independent regulons difficult within the scope of this work.
Overall, the data reveal a core FeHm modulon shared by all of the isolates studied. The core modulon includes genes in known regulons shown to have a role in virulence, such as the ArcA regulon . It is clear that the physiological roles of the genes in the putative core FeHm modulon are broad in the scope of function and may encompass most cellular processes including replication, energy metabolism, solute transport, protection against oxidation and biofilm formation. In addition, several new, previously uncharacterized genes have been identified with a possible role in FeHm metabolism. On the basis of our data it would appear that each of the three isolates may have a distinct set of non-core genes that respond to FeHm availability. Many of these genes are specific to individual isolates (see figure 4). While a common mechanism(s) for coordinated regulation of the core modulon across the species can be postulated, the regulation of the species-specific genes and strain-specific genes pose separate questions.
In summary, the major conclusions of this study are the following. First, the culture, microarray, and Q-PCR methodologies that we previously employed for the analysis of the regulation of gene expression by FeHm in the H. influenzae laboratory strain Rd KW20 were successfully extended to two recently sequenced clinical isolates, strains 10810 and R2866. Second, characterization of growth in vitro and conditions for reproducible transcriptional regulation by FeHm permitted direct comparison of gene expression among these three strains under identical environmental conditions. Third, among these three H. influenzae strains, a core set of genes responsive to environmental FeHm levels had been defined. The rigor and reproducibility of these microarray data were confirmed independently by Q-PCR. Fourth, these studies demonstrate the utility of comparative transcriptional profiling to identify genes shared across the species that are core members of an important modulon. These genes may play a key role in virulence; they are likely expressed during human disease; and thus, may comprise useful targets for potential therapeutic studies. These studies lay the foundation for further investigation of the role played by the FeHm modulon in H. influenzae virulence.