Discovery of intracellular heme-binding protein HrtR, which controls heme efflux by the conserved HrtB-HrtA transporter in Lactococcus lactis.

Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis.

Impact of aeration and heme-activated respiration on Lactococcus lactis gene expression: identification of a heme-responsive operon.

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O(2) and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.