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Zinc uptake in bacteria is essential to maintain cellular homeostasis and survival. ZnuABC is an important zinc importer of numerous bacterial genera, which is expressed to restore zinc homeostasis when the cytosolic concentration decreases beyond a critical threshold. Upon zinc limitation the fast-growing nonpathogenic organism Mycobacterium smegmatis (MSMEG) as well as the ruminant pathogen M. avium subsp. paratuberculosis (MAP) increases expression of genes encoding ZnuABC homologues, but also of genes encoding other transporters. This suggests an involvement of these transporters in zinc homeostasis. Here we characterized the putative zinc transporters of MSMEG (ZnuABC and ZnuABC2) and MAP (ZnuABC, MptABC, and MAP3774-76). Deletion of either ZnuABC or ZnuABC2 in MSMEG did not lead to growth defects, but to an increased expression of zinc marker genes in MSMEGΔznuABC, indicating cytosolic zinc limitation. However, chromatin immunoprecipitation proved direct binding of the global zinc regulator Zur to promoter regions of both znuABC and znuABC2. Simultaneous deletion of both transporters caused severe growth defects, which could be restored either by homologous complementation with single ZnuABC transporters or supplementation of growth media with zinc but not iron, manganese, cobalt, or magnesium. Heterologous complementation of the double mutant with MAP transporters also resulted in reconstitution of growth. Nonradioactive FluoZinTM-3AM zinc uptake assays directly revealed the competence of all transporters to import zinc. Finally, structural and phylogenetic analyses provided evidence of a novel class of ZnuABC transporters represented by the ZnuABC2 of MSMEG, which is present only in actinobacteria, mainly in the genera Nocardia, Streptomyces and fast growing Mycobacteria IMPORTANCEZinc is necessary for bacterial growth but simultaneously toxic when in excess. Hence, bacterial cells have developed systems to alter intracellular concentration. Regulation of these systems is primarily executed at transcriptional level by regulator proteins which sense femtomolar changes in the zinc level. In environmental and pathogenic mycobacteria zinc starvation induces expression of common zinc import systems such as the ZnuABC transporter, but also of other additional not yet characterized transport systems. In this study, we characterized the role of such systems in zinc transport. We showed that transport systems of both species whose transcription is induced upon zinc starvation can exchangeably restore cellular zinc homeostasis in transporter deficient mutants by transporting zinc into the cell.

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae. Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor delta-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.