Heme Uptake in Lactobacillus sakei Evidenced by a New Energy Coupling Factor (ECF)-Like Transport System.

Lactobacillus sakei is a nonpathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, although it possesses a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was previously shown by microscopy and the L. sakei genome reveals the complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (from lsa1836 to lsa1840 [lsa1836-1840]) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme-dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might correspond to a complete machinery of an energy coupling factor (ECF) transport system. We quantified in vitro the intracellular heme in the wild type (WT) and in our Δlsa1836-1840 deletion mutant using an intracellular heme sensor and inductively coupled plasma mass spectrometry for quantifying incorporated 57Fe heme. We showed that in the WT L. sakei, heme accumulation occurs rapidly and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. Therefore, this research outcome sheds new light on other possible functions of ECF-like systems.IMPORTANCELactobacillus sakei is a nonpathogenic bacterial species exhibiting high fitness in heme-rich environments such as meat products, although it does not need iron or heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for nonpathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei In this study, we demonstrate that one of them is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF; therefore, our study reveals a new class of heme transport system.

Enterococcal Rgg-like regulator ElrR activates expression of the elrA operon.

The Enterococcus faecalis leucine-rich protein ElrA promotes virulence by stimulating bacterial persistence in macrophages and production of the interleukin-6 (IL-6) cytokine. The ElrA protein is encoded within an operon that is poorly expressed under laboratory conditions but induced in vivo. In this study, we identify ef2687 (renamed elrR), which encodes a member of the Rgg (regulator gene for glucosyltransferase) family of putative regulatory proteins. Using quantitative reverse transcription-PCR, translational lacZ fusions, and electrophoretic mobility shift assays, we demonstrate that ElrR positively regulates expression of elrA. These results correlate with the attenuated virulence of the ΔelrR strain in a mouse peritonitis model. Virulence of simple and double elrR and elrA deletion mutants also suggests a remaining ElrR-independent expression of elrA in vivo and additional virulence-related genes controlled by ElrR.

Iron sources used by the nonpathogenic lactic acid bacterium Lactobacillus sakei as revealed by electron energy loss spectroscopy and secondary-ion mass spectrometry.

Lactobacillus sakei is a lactic acid bacterium naturally found on meat. Although it is generally acknowledged that lactic acid bacteria are rare species in the microbial world which do not have iron requirements, the genome sequence of L. sakei 23K has revealed quite complete genetic equipment dedicated to transport and use of this metal. Here, we aimed to investigate which iron sources could be used by this species as well as their role in the bacterium's physiology. Therefore, we developed a microscopy approach based on electron energy loss spectroscopy (EELS) analysis and nano-scale secondary-ion mass spectrometry (SIMS) in order to analyze the iron content of L. sakei cells. This revealed that L. sakei can use iron sources found in its natural ecosystem, myoglobin, hemoglobin, hematin, and transferrin, to ensure long-term survival during stationary phase. This study reveals that analytical image methods (EELS and SIMS) are powerful complementary tools for investigation of metal utilization by bacteria.