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J Bacteriol. Aug 2006; 188(15): 5640–5645.
PMCID: PMC1540045

Transcriptional Profile of Haemophilus influenzae: Effects of Iron and Heme

Abstract

Haemophilus influenzae requires either heme or a porphyrin and iron source for growth. Microarray studies of H. influenzae strain Rd KW20 identified 162 iron/heme-regulated genes, representing ~10% of the genome, with ≥1.5-fold changes in transcription in response to iron/heme availability in vitro. Eighty genes were preferentially expressed under iron/heme restriction; 82 genes were preferentially expressed under iron/heme-replete conditions.

While Haemophilus influenzae is unable to synthesize protoporphyrin IX (PPIX), it does possess a ferrochelatase that reversibly inserts iron into PPIX to form heme (13, 23). Thus, H. influenzae requires either iron, in the presence of PPIX, or heme to grow. Found only in humans, H. influenzae has evolved highly redundant mechanisms to obtain both heme and iron from host sources (16). The available heme/iron sources and the H. influenzae acquisition mechanisms have been partially characterized (6, 7, 14, 16-20, 26), although little is known about the specific regulation of these pathways.

The goals of the current study were to identify gene products potentially mediating iron and heme uptake and to provide a foundation for their further study, as well as to gain insight into possible mechanisms of regulation. Since H. influenzae can interconvert heme and iron (releasing PPIX), the genomic transcriptional profiles of growth in iron- and heme (FeHm)-replete and -restricted media were compared.

Initially the kinetics of FeHm-mediated derepression were characterized using quantitative reverse transcription-PCR (Q-RT-PCR) to examine transcripts of the FeHm-regulated iron binding protein gene hitA (1, 16). Inocula of H. influenzae Rd KW20 (ATCC 51907) were prepared as previously described (14). Inocula were added to 60-ml volumes of brain heart infusion (BHI) broth containing 10 μg/ml β-NAD (BHI-NAD) to yield ~2 × 107 CFU/ml. FeHm-replete cultures (containing 500 μM FeCl3 and 10 μg/ml heme as hemin chloride) and FeHm-restricted cultures (containing 150 μM deferoxamine) were incubated at 37°C in a rotary shaker (175 rpm). Samples were removed at 30-min intervals for Q-RT-PCR and viable count determination as previously described (15, 27). Cells remained viable upon transfer to FeHm-restricted BHI-NAD for at least 3 h (Fig. (Fig.1A),1A), and transcription of hitA increased to a plateau within 60 to 90 min of transfer to an FeHm-restricted environment (Fig. (Fig.1B).1B). Conversely, transcription of hitA in FeHm-supplemented media showed no increase over the same period.

FIG. 1.
Derepression and repression kinetics defining the window of transcriptional regulation for select FeHm-regulated genes of H. influenzae Rd KW20. (A) Growth kinetics (viable count) for the FeHm-replete (closed circles) and FeHm-restricted (open circles) ...

To determine the kinetics of FeHm repression, the experiment was repeated with supplementation of the FeHm-restricted media at 90 min with 500 μM FeCl3 and 10 μg/ml heme. Q-RT-PCR analysis demonstrated an initial increase in hitA transcripts; however, within 15 min of FeHm addition, transcripts rapidly decreased to a level identical to that in the FeHm-replete flask (Fig. (Fig.1C1C).

The transcription of tbp1 (11) and ompP2 (2) was also examined. These genes were considered to represent an iron/heme-regulated gene and a nonregulated gene, respectively. Genes hitA and tbp1 share a similar transcriptional profile, while ompP2 showed no changes in transcription (Fig. (Fig.1D).1D). Oligonucleotide primers are listed in Table S1 in the supplemental data at http://peds.ouhsc.edu/lab/stull/whitby2006/tableS1.pdf.

The kinetics of Fe repression and heme repression are similar.

The above experiment was repeated with addition of iron or heme alone. There were no apparent differences after supplementation with either iron or heme alone or with both in repression of transcription of hitA or tbp2 (Fig. (Fig.2);2); therefore, dual-iron/heme supplementation was utilized in further studies.

FIG. 2.
Comparison of the transcription change of the genes hitA (A) and tbp2 (B) following addition of iron (open circles), heme (closed triangles), or iron and heme (FeHm) together (open triangles) or with no addition (closed circles) following growth in FeHm-restricted ...

Identification of the FeHm regulon of H. influenzae.

The FeHm-restricted and FeHm-supplemented samples for microarray analysis were taken from a single flask, separated by 20 min. Three biological replicates of each condition were examined. The first sample was removed from FeHm-restricted media at 90 min postinoculation. Immediately, the flask was FeHm supplemented and a second sample was removed 20 min later. RNA for the microarray was prepared using Trizol (Invitrogen) as described by the manufacturer. Processing of the H. influenzae microarray was performed by Nimblegen (Madison, Wis.) following their protocols. Technical array replicates (arising from the presence of three arrays on each slide) were averaged prior to analysis of the biological replicates (8). The data were log2 transformed and compared between the two conditions by performing individual t tests using the TMEV software at http://www.tm4.org/mev.html (22). Genes with a 1.5-fold expression change and P < 0.05 were considered significant.

Of the 1,657 open reading frames on the Rd KW20 array, 162 were significantly differentially expressed (see Table S2 in the supplemental material at http://peds.ouhsc.edu/lab/stull/whitby2006/tableS2.pdf): 82 were maximally expressed under FeHm-replete conditions, and 80 were preferentially expressed under FeHm-restricted conditions. Comparison of microarray and Q-RT-PCR data for 11 genes indicated a linear correlation, with the microarray underestimating Q-RT-PCR values by approximately threefold.

FeHm-repressed transcripts contain genes with a known role in iron/heme uptake.

Of the 80 genes identified, 29 were single genes and 51 were in 21 putative operons. Analysis of the other genes in each operon indicated that while some genes had an increase in expression below the 1.5-fold threshold, all showed a statistically significant increase. Operons and stand-alone genes were determined based on a number of factors, including comparative genomics, transcriptional data from previous studies, conformity of orientation, size of intergenic regions, and prediction of putative transcriptional terminators using the program Transterm at http://cbcb.umb.edu/software/transterm (10). Included among the FeHm-repressible genes were known iron and heme acquisition genes (Table (Table1)1) including the hemoglobin/hemoglobin-haptoglobin receptor gene hgpC. (Strain Rd KW20 contains three potentially functional hgp genes, hgpBCD; however, both hgpB and hgpD are out of frame and not expressed.) To gain insight into possible mechanisms of regulation, we examined the upstream DNA for the presence of sequences associated with regulation by the ferric uptake regulator Fur (25). Potential sites were identified upstream of 32% of the FeHm-repressible operons.

TABLE 1.
H. influenzae Rd KW20 operons with altered transcription in response to environmental iron/heme levels

Identification of other genes with a putative role in iron/heme uptake.

Several additional genes encoding proteins with putative roles in iron transport were identified, including afuABC and yfeABCD and locus tags HI0663, HI0664, and HI1427. The afu locus shares homology with an Actinobacillus pleuropneumoniae ABC iron transporter (5). While the functions of HI1427, HI0663, and HI0664 are unknown, the putative product of HI1427 has characteristics consistent with periplasmic binding proteins, while the products of HI0663 and HI0664 both have characteristics of fused permease-ATPase components of ABC transporters. The yfe locus has been shown to mediate uptake of iron, manganese, and zinc in Yersinia pestis (3). Interestingly, transcription of a putative protease gene, pqqL (HI1368), was repressed by addition of FeHm. pqqL is in an operon encoding the TonB-dependent protein HI1369. Preliminary data indicate that HI1369 has a role in heme acquisition (data not shown). In other bacterial species, proteases play a direct role in the acquisition of iron and heme from various sources (12, 21, 24, 28).

The FeHm regulon contains genes with no known role in iron/heme uptake.

A number of genes preferentially expressed under FeHm limitation appear to have a direct role on cellular metabolism. These include genes encoding the enzymes phosphoenolpyruvate carboxykinase and fructose-1,6- bisphosphatase, suggesting that there may be a cellular shift to gluconeogenesis in the absence of FeHm. Consistent with this are the observations that genes encoding aspartate ammonia lyase, asparaginase B, malate dehydrogenase, and fumerase C are repressed by FeHm addition. Together, these enzymes would lead to the conversion of l-asparagine to phosphoenolpyruvate, which then feeds into gluconeogensis to generate glucose phosphate. The latter compound may then be shunted into glycogen by the action of the products of the malQ-glg locus, also preferentially expressed under FeHm limitation.

Identification of FeHm-induced genes.

Eighty-two genes were preferentially expressed under FeHm-replete conditions: of these 17 were stand-alone genes and 65 were contained within 25 putative operons (Table (Table1).1). Several of the identified genes have a role in iron and heme processing. For example, increased transcription of the bacterial ferritin genes (ftnA1 and ftnA2) would increase the ability to store and thus detoxify excess iron. Similarly, transcription of ccmDEFG (encoding a heme exporter involved in the biosynthesis of cytochrome c) is increased. Genes having a potential role in respiration display increased transcription. These include cydAB, encoding subunits of the terminal electron acceptor cytochrome d ubiquinol oxidase, and the nqr operon encoding an Na+-translocating NADH quinone oxidoreductase. This operon is similarly upregulated in Fe-rich environments in Neisseria gonorrhoeae (9). Other operons contain genes with iron-sulfur clusters (fdxH) or heme (fdxI) as cofactors.

Q-PCR of genes not represented on the microarray.

Two operons, which were repressed by FeHm, lacked several of the predicted genes on the array. HI0007 to HI0009 were shown, by microarray, to be upregulated upon the addition of FeHm. The genes represented by these three locus tags form a putative operon with HI0006m and together encode the subunits of the nitrate-inducible formate dehydrogenase. HI0006m was considered a pseudogene due to an internal stop codon and was not included on the microarray design. However, this stop codon is conserved in other bacteria, encoding a selenocysteine residue in the E. coli HI0006m homolog (4). Comparison of RNA levels of HI0006m and HI0007 using Q-PCR confirmed that HI0006m is also repressed by addition of FeHm, consistent with the other three genes of this operon.

The transcription of HI0343 and HI0345 (both excluded from the array) was compared to levels of HI0344. This analysis determined that the genes represented by these two locus tags are also upregulated by the addition of FeHm, consistent with the other genes in that operon (data not shown).

Conclusion.

The purpose of this study was to identify H. influenzae genes affected by iron and heme uptake. Defining the regulatory kinetics characterized a minimum window most likely to represent FeHm effects. Genes known to be involved in iron and heme uptake were clearly regulated in a synchronous manner. Furthermore, significant changes in transcription of all genes in the regulated operons were demonstrated. The identification of FeHm-regulated protease and potential periplasmic transport systems provides targets for future studies. Since iron and heme are sequestered in vivo, it is likely that the pattern of regulation observed in vitro relates to the transcriptional status during infection, supporting previous studies demonstrating transcription of FeHm-repressible genes during otitis media (29).

Microarray data accession number.

Microarray data have been deposited with the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE5061.

Acknowledgments

This work was supported by Public Health Service grant AI29611 from the National Institutes of Allergy and Infectious Diseases to T.L.S., D.J.M., and P.W.W. We gratefully acknowledge the support of the Children's Medical Research Institute.

We thank Jennifer Springer for technical assistance.

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