The Bacillus subtilis yqgC-sodA operon protects magnesium-dependent enzymes by supporting manganese efflux.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function, and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations, they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the YS deletion mutants results from distinct enzymatic vulnerabilities.IMPORTANCEBacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. In Bacillus subtilis, Mn-dependent superoxide dismutase (MnSOD) is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual proteins) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes.

Manganese impairs the QoxABCD terminal oxidase leading to respiration-associated toxicity.

Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown. Here, we employed a forward genetics approach to identify pathways affected by manganese intoxication. Our results highlight a central role for the membrane-localized electron transport chain in metal intoxication during aerobic growth. In the presence of elevated manganese, there is an increased generation of reactive radical species associated with dysfunction of the major terminal oxidase, the cytochrome aa3 heme-copper menaquinol oxidase (QoxABCD). Intoxication is suppressed by diversion of menaquinol to alternative oxidases or by a mutation affecting heme A synthesis that is known to convert QoxABCD from an aa3 to a bo3 -type oxidase. Manganese sensitivity is also reduced by derepression of the MhqR regulon, which protects cells against reactive quinones. These results suggest that dysfunction of the cytochrome aa3 -type quinol oxidase contributes to metal-induced intoxication.

Mutations of the Bacillus subtilis YidC1 (SpoIIIJ) insertase alleviate stress associated with σM-dependent membrane protein overproduction.

In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 (spoIIIJ), which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high σM. Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.

Heme Binding to HupZ with a C-Terminal Tag from Group A Streptococcus.

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV-vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

The GAS PefCD exporter is a MDR system that confers resistance to heme and structurally diverse compounds.

BACKGROUND: Group A streptococcus (GAS) is the etiological agent of a variety of local and invasive infections as well as post-infection complications in humans. This ß-hemolytic bacterium encounters environmental heme in vivo in a concentration that depends on the infection type and stage. While heme is a noxious molecule, the regulation of cellular heme levels and toxicity is underappreciated in GAS. We previously reported that heme induces three GAS genes that are similar to the pefRCD (porphyrin regulated efflux) genes from group B streptococcus. Here, we investigate the contributions of the GAS pef genes to heme management and physiology. RESULTS: In silico analysis revealed that the PefCD proteins entail a Class-1 ABC-type transporter with homology to selected MDR systems from Gram-positive bacteria. RT-PCR experiments confirmed that the pefRCD genes are transcribed to polycistronic mRNA and that a pefC insertion inactivation mutant lost the expression of both pefC and pefD genes. This mutant was hypersensitive to heme, exhibiting significant growth inhibition already in the presence of 1 µM heme. In addition, the pefC mutant was more sensitive to several drugs and nucleic acid dyes and demonstrated higher cellular accumulation of heme in comparison with the wild type and the complemented strains. Finally, the absence of the PefCD transporter potentiated the damaging effects of heme on GAS building blocks including lipids and DNA. CONCLUSION: We show here that in GAS, the pefCD genes encode a multi-drug efflux system that allows the bacterium to circumvent the challenges imposed by labile heme. This is the first heme resistance machinery described in GAS.

In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus.

In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split ß-barrel conformation in each monomer comprising six ß strands and three α helices. This structure resembles the split ß-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split ß-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.

Structural basis for transcription activation through cooperative recruitment of MntR.

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn 2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn 2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

The Bacillus subtilis yqgC-sodA operon protects magnesium-dependent enzymes by supporting manganese efflux.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the yqgC-sodA deletion mutants results from distinct enzymatic vulnerabilities.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn 2+ -dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn 2+ -dependent membrane and extracellular enzymes.

TerC proteins function during protein secretion to metalate exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

Resource sharing between central metabolism and cell envelope synthesis.

Synthesis of the bacterial cell envelope requires a regulated partitioning of resources from central metabolism. Here, we consider the key metabolic junctions that provide the precursors needed to assemble the cell envelope. Peptidoglycan synthesis requires redirection of a glycolytic intermediate, fructose-6-phosphate, into aminosugar biosynthesis by the highly regulated branchpoint enzyme GlmS. MurA directs the downstream product, UDP-GlcNAc, specifically into peptidoglycan synthesis. Other shared resources required for cell envelope synthesis include the isoprenoid carrier lipid undecaprenyl phosphate and amino acids required for peptidoglycan cross-bridges. Assembly of the envelope requires a sharing of limited resources between competing cellular pathways and may additionally benefit from scavenging of metabolites released from neighboring cells or the formation of symbiotic relationships with a host.

A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.

A bacterial checkpoint protein for ribosome assembly moonlights as an essential metabolite-proofreading enzyme.

In eukaryotes, adventitious oxidation of erythrose-4-phosphate, an intermediate of the pentose phosphate pathway (PPP), generates 4-phosphoerythronate (4PE), which inhibits 6-phosphogluconate dehydrogenase. 4PE is detoxified by metabolite-proofreading phosphatases such as yeast Pho13. Here, we report that a similar function is carried out in Bacillus subtilis by CpgA, a checkpoint protein known to be important for ribosome assembly, cell morphology and resistance to cell wall-targeting antibiotics. We find that ΔcpgA cells are intoxicated by glucose or other carbon sources that feed into the PPP, and that CpgA has high phosphatase activity with 4PE. Inhibition of 6-phosphogluconate dehydrogenase (GndA) leads to intoxication by 6-phosphogluconate, a potent inhibitor of phosphoglucose isomerase (PGI). The coordinated shutdown of PPP and glycolysis leads to metabolic gridlock. Overexpression of GndA, PGI, or yeast Pho13 suppresses glucose intoxication of ΔcpgA cells, but not cold sensitivity, a phenotype associated with ribosome assembly defects. Our results suggest that CpgA is a multifunctional protein, with genetically separable roles in ribosome assembly and metabolite proofreading.

The crimson conundrum: heme toxicity and tolerance in GAS.

The massive erythrocyte lysis caused by the Group A Streptococcus (GAS) suggests that the ß-hemolytic pathogen is likely to encounter free heme during the course of infection. In this study, we investigated GAS mechanisms for heme sensing and tolerance. We compared the minimal inhibitory concentration of heme among several isolates and established that excess heme is bacteriostatic and exposure to sub-lethal concentrations of heme resulted in noticeable damage to membrane lipids and proteins. Pre-exposure of the bacteria to 0.1 µM heme shortened the extended lag period that is otherwise observed when naive cells are inoculated into heme-containing medium, implying that GAS is able to adapt. The global response to heme exposure was determined using microarray analysis revealing a significant transcriptome shift that included 79 up regulated and 84 down regulated genes. Among other changes, the induction of stress-related chaperones and proteases, including groEL/ES (8x), the stress regulators spxA2 (5x) and ctsR (3x), as well as redox active enzymes were prominent. The heme stimulon also encompassed a number of regulatory proteins and two-component systems that are important for virulence. A three-gene cluster that is homologous to the pefRCD system of the Group B Streptococcus was also induced by heme. PefR, a MarR-like regulator, specifically binds heme with stoichiometry of 1:2 and protoporphyrin IX (PPIX) with stoichiometry of 1:1, implicating it is one of the GAS mediators to heme response. In summary, here we provide evidence that heme induces a broad stress response in GAS, and that its success as a pathogen relies on mechanisms for heme sensing, detoxification, and repair.