Bacterial TANGO2 homologs are heme-trafficking proteins that facilitate biosynthesis of cytochromes c.

Heme, an essential molecule for virtually all living organisms, acts primarily as a cofactor in a large number of proteins. However, how heme is mobilized from the site of synthesis to the locations where hemoproteins are assembled remains largely unknown in cells, especially bacterial ones. In this study, with Shewanella oneidensis as the model, we identified HtpA (SO0126) as a heme-trafficking protein and homolog of TANGO2 proteins found in eukaryotes. We showed that HtpA homologs are widely distributed in all domains of living organisms and have undergone parallel evolution. In its absence, the cytochrome (cyt) c content and catalase activity decreased significantly. We further showed that both HtpA and representative TANGO2 proteins bind heme with 1:1 stoichiometry and a relatively low dissociation constant. Protein interaction analyses substantiated that HtpA directly interacts with the cytochrome c maturation system. Our findings shed light on cross-membrane transport of heme in bacteria and extend the understanding of TANGO2 proteins. IMPORTANCE The intracellular trafficking of heme, an essential cofactor for hemoproteins, remains underexplored even in eukaryotes, let alone bacteria. Here we developed a high-throughput method by which HtpA, a homolog of eukaryotic TANGO2 proteins, was identified to be a heme-binding protein that enhances cytochrome c biosynthesis and catalase activity in Shewanella oneidensis. HtpA interacts with the cytochrome c biosynthesis system directly, supporting that this protein, like TANGO2, functions in intracellular heme trafficking. HtpA homologs are widely distributed, but a large majority of them were found to be non-exchangeable, likely a result of parallel evolution. By substantiating the heme-trafficking nature of HtpA and its eukaryotic homologs, our findings provide general insight into the heme-trafficking process and highlight the functional conservation along evolution in all living organisms.

Efficacy of 405-nm LED illumination and citral used alone and in combination for the inactivation of Cronobacter sakazakii in reconstituted powdered infant formula.

Cronobacter sakazakii, a foodborne opportunistic pathogen, mainly affects neonates and infants, with mortality rates of 26.9%. Most outbreaks arise from powdered infant formula (PIF). The aim of this study was to investigate the efficacy and mechanism of 405-nm light-emitting diode (LED) and citral treatment used in combination against C. sakazakii in reconstituted PIF. LED-illumination combined with citral showed better antimicrobial effects than either treatment alone. In reconstituted PIF, the abundance of C. sakazakii cells was reduced by 6.5 log 10 CFU/mL following combined LED and 9 µL/mL citral treatment for 90 min compared with untreated controls, respectively. Combined LED and 6 µL/mL citral treatment destroyed C. sakazakii cellular morphology and membrane integrity, prolonged the recovery time of sublethally-injured cells, and induced lipid peroxidation. Besides, LED treatment decreased the amount of lipid peroxidation caused by citral treatment alone. Neither LED illumination nor citral treatment resulted in breakdown of C. sakazakii genomic DNA. Because of its safe, environmentally-friendly, economical, and high-performance characteristics, the combination of LED-illumination and citral treatment has the potential to be developed into a strategy to control C. sakazakii contamination in stored and reconstituted PIF.

NapB Restores cytochrome c biosynthesis in bacterial dsbD-deficient mutants.

Cytochromes c (cyts c), essential for respiration and photosynthesis in eukaryotes, confer bacteria respiratory versatility for survival and growth in natural environments. In bacteria having a cyt c maturation (CCM) system, DsbD is required to mediate electron transport from the cytoplasm to CcmG of the Ccm apparatus. Here with cyt c-rich Shewanella oneidensis as the research model, we identify NapB, a cyt c per se, that suppresses the CCM defect of a dsbD mutant during anaerobiosis, when NapB is produced at elevated levels, a result of activation by cAMP-Crp. Data are then presented to suggest that NapB reduces CcmG, leading to the suppression. We further show that NapB proteins capable of rescuing CCM in the dsbD mutant form a small distinct clade. The study sheds light on multifunctionality of cyts c, and more importantly, unravels a self-salvation strategy through which bacteria have evolved to better adjust to the natural world.

Physiological Roles of Nitrite and Nitric Oxide in Bacteria: Similar Consequences from Distinct Cell Targets, Protection, and Sensing Systems.

Nitrite and nitric oxide (NO) are two active nitrogen oxides that display similar biochemical properties, especially when interacting with redox-sensitive proteins (i.e., hemoproteins), an observation serving as the foundation of the notion that the antibacterial effect of nitrite is largely attributed to NO formation. However, a growing body of evidence suggests that they are largely treated as distinct molecules by bacterial cells. Although both nitrite and NO are formed and decomposed by enzymes participating in the transformation of these nitrogen species, NO can also be generated via amino acid metabolism by bacterial NO synthetase and scavenged by flavohemoglobin. NO seemingly interacts with all hemoproteins indiscriminately, whereas nitrite shows high specificity to heme-copper oxidases. Consequently, the homeostasis of redox-sensitive proteins may be responsible for the substantial difference in NO-targets identified to date among different bacteria. In addition, most protective systems against NO damage have no significant role in alleviating inhibitory effects of nitrite. Furthermore, when functioning as signal molecules, nitrite and NO are perceived by completely different sensing systems, through which they are linked to different biological processes.

Plasticity of the peroxidase AhpC links multiple substrates to diverse disulfide-reducing pathways in Shewanella oneidensis.

AhpC is a bacterial representative of 2-Cys peroxiredoxins (Prxs) with broad substrate specificity and functional plasticity. However, details underpinning these two important attributes of AhpC remain unclear. Here, we studied the functions and mechanisms of regulation of AhpC in the facultative Gram-negative anaerobic bacterium Shewanella oneidensis, in which AhpC's physiological roles can be conveniently assessed through its suppression of a plating defect due to the genetic loss of a major catalase. We show that successful suppression can be achieved only when AhpC is produced in a dose- and time-dependent manner through a complex mechanism involving activation of the transcriptional regulator OxyR, transcription attenuation, and translation reduction. By analyzing AhpC truncation variants, we demonstrate that reactivity with organic peroxides (OPs) rather than H2O2 is resilient to mutagenesis, implying that OP reduction is the core catalytic function of AhpC. Intact AhpC could be recycled only by its cognate reductase AhpF, and AhpC variants lacking the Prx domain or the extreme C-terminal five residues became promiscuous electron acceptors from the thioredoxin reductase TrxR and the GSH reductase Gor in addition to AhpF, implicating an additional dimension to functional plasticity of AhpC. Finally, we show that the activity of S. oneidensis AhpC is less affected by mutations than that of its Escherichia coli counterpart. These findings suggest that the physiological roles of bacterial AhpCs are adapted to different oxidative challenges, depending on the organism, and that its functional plasticity is even more extensive than previously reported.

Nitrite modulates aminoglycoside tolerance by inhibiting cytochrome heme-copper oxidase in bacteria.

As a bacteriostatic agent, nitrite has been used in food preservation for centuries. When used in combination with antibiotics, nitrite is reported to work either cooperatively or antagonistically. However, the mechanism underlying these effects remains largely unknown. Here we show that nitrite mediates tolerance to aminoglycosides in both Gram-negative and Gram-positive bacteria, but has little interaction with other types of antibiotics. Nitrite directly and mainly inhibits cytochrome heme-copper oxidases (HCOs), and by doing so, the membrane potential is compromised, blocking uptake of aminoglycosides. In contrast, reduced respiration (oxygen consumption rate) resulting from nitrite inhibition is not critical for aminoglycoside tolerance. While our data indicate that nitrite is a promising antimicrobial agent targeting HCOs, cautions should be taken when used with other antibiotics, aminoglycosides in particular.

Complex Oxidation of Apocytochromes c during Bacterial Cytochrome c Maturation.

c-Type cytochromes (cyts c) are proteins that contain covalently bound heme and that thus require posttranslational modification for activity, a process carried out by the cytochrome c (cyt c) maturation system (referred to as the Ccm system) in many Gram-negative bacteria. It has been established that during cyt c maturation (CCM), two cysteine thiols of the heme binding motif (CXXCH) within apocytochromes c (apocyts c) are first oxidized largely by DsbA to form a disulfide bond, which is later reduced through a thio-reductive pathway involving DsbD. However, the physiological impacts of DsbA proteins on CCM in fact vary significantly among bacteria. In this work, we used the cyt c-rich Gram-negative bacterium Shewanella oneidensis as the research model to clarify the roles of DsbA proteins in CCM. We show that in terms of the oxidation of apocyts c, DsbA proteins are an important but not critical factor, and, strikingly, oxygen is not either. By exploiting the DsbD-independent pathway, we identify DsbA1, DsbA2, and DsbA3 as oxidants contributing to the oxidation of apocyts c and reductants, such as cysteine, to be an effective antagonist against DsbA-independent oxidation. We further show that DsbB proteins are partially responsible for the reoxidization of reduced DsbA proteins. Overall, our results indicate that the DsbA-DsbB redox pair has a limited role in CCM, challenging the established notion that it is the main oxidant for apocyts cIMPORTANCE DsbA is a powerful oxidase that functions in the bacterial periplasm to introduce disulfide bonds in many proteins, including apocytochromes c It has been well established that although DsbA is not essential, it plays a primary role in cytochrome c maturation, based on studies in bacteria hosting several cyts c Here, with cyt c-rich S. oneidensis as a research model, we show that this is not always the case. Moreover, we demonstrate that DsbB is also not essential for cytochrome c maturation. These results underscore the need to identify oxidants other than DsbA/DsbB that are crucial in the oxidation of apocyts c in bacteria.