Activator of one protease transforms into inhibitor of another in response to nutritional signals.

All cells use proteases to adjust protein amounts. Proteases maintain protein homeostasis by degrading nonfunctional toxic proteins and play regulatory roles by targeting particular substrates in response to specific signals. Here we address how cells tune protease specificity to nutritional signals. We report that Salmonella enterica increases the specificity of the broadly conserved proteases Lon and ClpSAP by transforming the Lon activator and substrate HspQ into an inhibitor of the N-degron recognin ClpS, the adaptor of the ClpAP protease. We establish that upon acetylation, HspQ stops being a Lon activator and substrate and that the accumulated HspQ binds to ClpS, hindering degradation of ClpSAP substrates. Growth on glucose promotes HspQ acetylation by increasing acetyl-CoA amounts, thereby linking metabolism to proteolysis. By altering protease specificities but continuing to degrade junk proteins, cells modify the abundance of particular proteins while preserving the quality of their proteomes. This rapid response mechanism linking protease specificity to nutritional signals is broadly conserved.

Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate.

According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP, as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.

Reducing Ribosome Biosynthesis Promotes Translation during Low Mg2+ Stress.

The synthesis of ribosomes is regulated by both amino acid abundance and the availability of ATP, which regenerates guanosine triphosphate (GTP), powers ribosomes, and promotes transcription of rRNA genes. We now report that bacteria supersede both of these controls when experiencing low cytosolic magnesium (Mg2+), a divalent cation essential for ribosome stabilization and for neutralization of ATP's negative charge. We uncover a regulatory circuit that responds to low cytosolic Mg2+ by promoting expression of proteins that import Mg2+ and lower ATP amounts. This response reduces the levels of ATP and ribosomes, making Mg2+ ions available for translation. Mutants defective in Mg2+ uptake and unable to reduce ATP levels accumulate non-functional ribosomal components and undergo translational arrest. Our findings establish a paradigm whereby cells reduce the amounts of translating ribosomes to carry out protein synthesis.

Small proteins regulate Salmonella survival inside macrophages by controlling degradation of a magnesium transporter.

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1+/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.

A protein that controls the onset of a Salmonella virulence program.

The mechanism of action and contribution to pathogenesis of many virulence genes are understood. By contrast, little is known about anti-virulence genes, which contribute to the start, progression, and outcome of an infection. We now report how an anti-virulence factor in Salmonella enterica serovar Typhimurium dictates the onset of a genetic program that governs metabolic adaptations and pathogen survival in host tissues. Specifically, we establish that the anti-virulence protein CigR directly restrains the virulence protein MgtC, thereby hindering intramacrophage survival, inhibition of ATP synthesis, stabilization of cytoplasmic pH, and gene transcription by the master virulence regulator PhoP. We determine that, like MgtC, CigR localizes to the bacterial inner membrane and that its C-terminal domain is critical for inhibition of MgtC. As in many toxin/anti-toxin genes implicated in antibiotic tolerance, the mgtC and cigR genes are part of the same mRNA. However, cigR is also transcribed from a constitutive promoter, thereby creating a threshold of CigR protein that the inducible MgtC protein must overcome to initiate a virulence program critical for pathogen persistence in host tissues.

The expanded specificity and physiological role of a widespread N-degron recognin.

All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacterium Salmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

Low Cytoplasmic Magnesium Increases the Specificity of the Lon and ClpAP Proteases.

Proteolysis is a fundamental property of all living cells. In the bacterium Salmonella enterica serovar Typhimurium, the HspQ protein controls the specificities of the Lon and ClpAP proteases. Upon acetylation, HspQ stops being a Lon substrate and no longer enhances proteolysis of the Lon substrate Hha. The accumulated HspQ protein binds to the protease adaptor ClpS, hindering proteolysis of ClpS-dependent substrates of ClpAP, such as Oat, a promoter of antibiotic persistence. HspQ is acetylated by the protein acetyltransferase Pat from acetyl coenzyme A (acetyl-CoA) bound to the acetyl-CoA binding protein Qad. We now report that low cytoplasmic Mg2+ promotes qad expression, which protects substrates of Lon and ClpSAP by increasing HspQ amounts. The qad promoter is activated by PhoP, a regulatory protein highly activated in low cytoplasmic Mg2+ that also represses clpS transcription. Both the qad gene and PhoP repression of the clpS promoter are necessary for antibiotic persistence. PhoP also promotes qad transcription in Escherichia coli, which shares a similar PhoP box in the qad promoter region with S. Typhimurium, Salmonella bongori, and Enterobacter cloacae. Our findings identify cytoplasmic Mg2+ and the PhoP protein as critical regulators of protease specificity in multiple enteric bacteria. IMPORTANCE The bacterium Salmonella enterica serovar Typhimurium narrows down the spectrum of substrates degraded by the proteases Lon and ClpAP in response to low cytoplasmic Mg2+, a condition that decreases protein synthesis. This control is exerted by PhoP, a transcriptional regulator activated in low cytoplasmic Mg2+ that governs proteostasis and is conserved in enteric bacteria. The uncovered mechanism enables bacteria to control the abundance of preexisting proteins.

Reduction in adaptor amounts establishes degradation hierarchy among protease substrates.

ATP-dependent proteases control critical cellular processes, including development, physiology, and virulence. A given protease may recognize a substrate directly via an unfoldase domain or subunit or indirectly via an adaptor that delivers the substrate to the unfoldase. We now report that cells achieve differential stability among substrates of a given protease by modulating adaptor amounts. We establish that the regulatory protein PhoP represses transcription of the gene specifying the ClpAP protease adaptor ClpS when the bacteria Salmonella enterica and Escherichia coli experience low cytoplasmic Mg2+ The resulting decrease in ClpS amounts diminishes proteolysis of several ClpSAP-dependent substrates, including the putrescine aminotransferase Oat, which heightens the formation of antibiotic persisters, and the transcriptional regulators UvrY and PhoP, which alter the expression of genes controlled by these proteins. By contrast, the decrease in ClpS amounts did not impact the abundance of the ClpSAP substrate FtsA, reflecting that FtsA binds to ClpS more tightly than to UvrY and PhoP. Our findings show how physiological conditions that reduce adaptor amounts modify the abundance of selected substrates of a given protease.

The Microbiological Characteristics of Acinetobacter Baumannii Associated With Early Mortality in Patients With Bloodstream Infection.

Background: Despite rapid deaths resulting from Acinetobacter baumannii bacteremia, the clinical impact of the microbiological characteristics of A baumannii strains on early mortality (EM) is unclear. We aimed to identify the microbiological characteristics of A baumannii strains associated with EM. Methods: Clinical information and isolates from patients with A baumannii bacteremia from January 2015 to December 2021 were collected. EM was defined as death within 3 days of the initial positive blood culture, whereas late mortality meant death within 5-30 days. The microbiological characteristics of A baumannii were analyzed using multilocus sequence typing, polymerase chain reactions, and a Galleria mellonella in vivo infection model. Results: Among 130 patients, 69 (53.1%) died within 30 days and EM occurred in 38 (55.1% of 30-day deaths). Sequence type 191 (ST191) strain was more prevalent in patients with EM than in 30-day survivors (31.6% vs 6.6%). Regarding virulence genes, bfmS was more frequent (92.1% vs 47.5%), whereas bauA was less frequent (13.2% vs 52.5%) in patients with EM than in 30-day survivors. Higher clinical severity, pneumonia, and ST191 infection were identified as independent risk factors for EM. In the G mellonella infection model, ST191, bfmS+, and bauA- isolates showed higher virulence than non-ST191, bfmS-, and bauA+ isolates, respectively. Conclusions: ST191 and bfmS were more frequently found in the EM group. ST191 infection was also an independent risk factor for EM and highly virulent in the in vivo model. Tailored infection control measures based on these characteristics are necessary for A baumannii bacteremia management.

Staphylococcus argenteus bacteremia in the Republic of Korea.

In 2015, Staphylococcus argenteus and Staphylococcus schweitzeri were proposed as new species, distinct from Staphylococcus aureus and collectively referred to as the S. aureus complex. However, no clinical reports of these new species exist in Korea. Upon the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for all bloodstream isolates since September 2022, S. argenteus was identified in one patient. Therefore, we aimed to search for new species among the archives of the S. aureus bacteremia cohort and describe their clinical and microbiological characteristics. Among the 691 archived S. aureus isolates between 2012 and 2018, one was identified as S. argenteus via MALDI-TOF MS. Both S. argenteus isolates (one in 2022) were obtained from patients with extensive pneumonia accompanied by bacteremia and both cases had fatal outcomes. They harbored multiple virulence genes (clfA, clfB, fnbpA, sdrC, sdrD, sdrE, bbp, cna, see, seg, sei, blaZ, fnbpB, and map) but did not harbor mecA and pvl. No matched sequence type (ST) was found in either isolate, and both S. argenteus isolates were closely related to ST1594, ST1593, ST1793, and ST1303, which belonged to S. argenteus. S. argenteus accounted for <1% of the S. aureus complex but had clinical characteristics similar to S. aureus. Therefore, clinicians should be aware of these factors to avoid misidentifying these strains as coagulase-negative staphylococci, and appropriate reporting is required to minimize confusion.IMPORTANCEStaphylococcus argenteus, a member of Staphylococcus aureus complex, has been reported as an important pathogen that causes clinically invasive infections in humans similar to S. aureus. Clinical isolates of S. argenteus have been reported across the world, showing a large geographical difference in prevalence and genomic profile. However, there have been no clinical reports regarding this new species in Korea. This is the first report to investigate the clinical and genetic characteristics of S. argenteus identified in patients with bacteremia, and the proportion of S. argenteus bacteremia among S. aureus bacteremia cohort in Korea.

ATP-dependent RecG helicase is required for the transcriptional regulator OxyR function in Pseudomonas species.

The oxyR gene appears to reside in an operon with the recG helicase gene in many bacteria, including pathogenic Pseudomonas aeruginosa and Pseudomonas putida. Analysis of P. putida transcriptomes shows that many OxyR-controlled genes are regulated by the ATP-dependent RecG helicase and that RecG alone modulates the expression of many genes. We found that purified RecG binds to the promoters of many OxyR-controlled genes and that expression of these genes was not induced under conditions of oxidative stress in recG mutants of P. aeruginosa, P. putida, and Escherichia coli. In vitro data revealed that promoters containing palindromic sequences are essential for RecG binding and that single-strand binding proteins and ATP are also needed for RecG to promote transcription, whereas a magnesium ion has the opposite effect. The OxyR tetramer preferentially binds to promoters after RecG has generated linear DNA in the presence of ATP; otherwise, the OxyR dimer has higher affinity. This study provides new insights into the mechanism of bacterial transcription by demonstrating that RecG might be required for the induction of the OxyR regulon by unwinding palindromic DNA for transcription. This work describes a novel bacterial transcriptional function by RecG helicase with OxyR and may provide new targets for controlling Pseudomonas species pathogen.

Membrane Proteins as a Regulator for Antibiotic Persistence in Gram-Negative Bacteria.

Antibiotic treatment failure threatens our ability to control bacterial infections that can cause chronic diseases. Persister bacteria are a subpopulation of physiological variants that becomes highly tolerant to antibiotics. Membrane proteins play crucial roles in all living organisms to regulate cellular physiology. Although a diverse membrane component involved in persistence can result in antibiotic treatment failure, the regulations of antibiotic persistence by membrane proteins has not been fully understood. In this review, we summarize the recent advances in our understanding with regards to membrane proteins in Gram-negative bacteria as a regulator for antibiotic persistence, highlighting various physiological mechanisms in bacteria.

Chemical modulation of SQSTM1/p62-mediated xenophagy that targets a broad range of pathogenic bacteria.

The N-degron pathway is a proteolytic system in which the N-terminal degrons (N-degrons) of proteins, such as arginine (Nt-Arg), induce the degradation of proteins and subcellular organelles via the ubiquitin-proteasome system (UPS) or macroautophagy/autophagy-lysosome system (hereafter autophagy). Here, we developed the chemical mimics of the N-degron Nt-Arg as a pharmaceutical means to induce targeted degradation of intracellular bacteria via autophagy, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Escherichia coli, and Streptococcus pyogenes as well as Mycobacterium tuberculosis (Mtb). Upon binding the ZZ domain of the autophagic cargo receptor SQSTM1/p62 (sequestosome 1), these chemicals induced the biogenesis and recruitment of autophagic membranes to intracellular bacteria via SQSTM1, leading to lysosomal degradation. The antimicrobial efficacy was independent of rapamycin-modulated core autophagic pathways and synergistic with the reduced production of inflammatory cytokines. In mice, these drugs exhibited antimicrobial efficacy for S. Typhimurium, Bacillus Calmette-Guérin (BCG), and Mtb as well as multidrug-resistant Mtb and inhibited the production of inflammatory cytokines. This dual mode of action in xenophagy and inflammation significantly protected mice from inflammatory lesions in the lungs and other tissues caused by all the tested bacterial strains. Our results suggest that the N-degron pathway provides a therapeutic target in host-directed therapeutics for a broad range of drug-resistant intracellular pathogens.Abbreviations: ATG: autophagy-related gene; BCG: Bacillus Calmette-Guérin; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CFUs: colony-forming units; CXCL: C-X-C motif chemokine ligand; EGFP: enhanced green fluorescent protein; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PB1: Phox and Bem1; SQSTM1/p62: sequestosome 1; S. Typhimurium: Salmonella enterica serovar Typhimurium; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; UBA: ubiquitin-associated.

Iron homeostasis affects antibiotic-mediated cell death in Pseudomonas species.

Antibiotics can induce cell death via a variety of action modes, including the inhibition of transcription, ribosomal function, and cell wall biosynthesis. In this study, we demonstrated directly that iron availability is important to the action of antibiotics, and the ferric reductases of Pseudomonas putida and Pseudomonas aeruginosa could accelerate antibiotic-mediated cell death by promoting the Fenton reaction. The modulation of reduced nicotinamide-adenine dinucleotide (NADH) levels and iron chelation affected the actions of antibiotics. Interestingly, the deletion of the ferric reductase gene confers more antibiotic resistance upon cells, and its overexpression accelerates antibiotic-mediated cell death. The results of transcriptome analysis showed that both Pseudomonas species induce many oxidative stress genes under antibiotic conditions, which could not be observed in ferric reductase mutants. Our results indicate that iron homeostasis is crucial for bacterial cell survival under antibiotics and should constitute a significant target for boosting the action of antibiotics.

Reduced ATP-dependent proteolysis of functional proteins during nutrient limitation speeds the return of microbes to a growth state.

When cells run out of nutrients, the growth rate greatly decreases. Here, we report that microorganisms, such as the bacterium Salmonella enterica serovar Typhimurium, speed up the return to a rapid growth state by preventing the proteolysis of functional proteins by ATP-dependent proteases while in the slow-growth state or stationary phase. This reduction in functional protein degradation resulted from a decrease in the intracellular concentration of ATP that was nonetheless sufficient to allow the continued degradation of nonfunctional proteins by the same proteases. Protein preservation occurred under limiting magnesium, carbon, or nitrogen conditions, indicating that this response was not specific to low availability of a particular nutrient. Nevertheless, the return to rapid growth required proteins that mediate responses to the specific nutrient limitation conditions, because the transcriptional regulator PhoP was necessary for rapid recovery only after magnesium starvation. Reductions in intracellular ATP and in ATP-dependent proteolysis also enabled the yeast Saccharomyces cerevisiae to recover faster from stationary phase. Our findings suggest that protein preservation during a slow-growth state is a conserved microbial strategy that facilitates the return to a growth state once nutrients become available.

Molecular characterization of FinR, a novel redox-sensing transcriptional regulator in Pseudomonas putida KT2440.

FinR is required for the induction of fpr (ferredoxin-NADP(+) reductase) under superoxide stress conditions in Pseudomonas putida. Many proteobacteria harbour FinR homologues in their genome as a putative LysR-type protein. Three cysteine residues (at positions 150, 239 and 289 in P. putida FinR) are conserved in all FinR homologues. When these conserved cysteines, along with two other cysteine residues present in FinR, were individually mutated to serines, the FinR remained active, unlike SoxR and OxyR in Escherichia coli. The results of our in vitro DNA-binding assay with cellular extracts showed that FinR binds directly to the fpr promoter region. In order to identify the FinR functional domain for sensing superoxide stress, we employed random and site-directed mutagenesis of FinR. Among 18 single amino acid mutants, three mutants (T39A, R194A and E225A) abolished fpr induction without any alteration of their DNA-binding ability, whereas other mutants also abrogated their DNA-binding abilities. Interestingly, two mutants (L215P and D51A) appeared to be constitutively active, regardless of superoxide stress conditions. Ferrous iron depletion, ferric iron addition and fdxA (ferredoxin) gene deletion also participate in the regulation of fpr. These data indicate that FinR has unusual residues for redox sensing and that the redox-sensing mechanism of FinR differs from the well-known mechanisms of OxyR and SoxR.

Phenotypic and physiological alterations by heterologous acylhomoserine lactone synthase expression in Pseudomonas putida.

Many bacteria harbour an incomplete quorum-sensing (QS) system, whereby they possess LuxR homologues without the QS acylhomoserine lactone (AHL) synthase, which is encoded by a luxI homologue. An artificial AHL-producing plasmid was constructed using a cviI gene encoding the C6-AHL [N-hexanoyl homoserine lactone (HHL)] synthase from Chromobacterium violaceum, and was introduced successfully into both the wild-type and a ppoR (luxR homologue) mutant of Pseudomonas putida. Our data provide evidence to suggest that the PpoR-HHL complex, but neither PpoR nor HHL alone, could attenuate growth, antibiotic resistance and biofilm formation ability. In contrast, swimming motility, siderophore production and indole degradation were enhanced by PpoR-HHL. The addition of exogenous indole increased biofilm formation and reduced swimming motility. Interestingly, indole proved ineffective in the presence of PpoR-HHL, thereby suggesting that the PpoR-HHL complex masks the effects of indole. Our data were supported by transcriptome analyses, which showed that the presence of the plasmid-encoded AHL synthase altered the expression of many genes on the chromosome in strain KT2440. Our results showed that heterologous luxI expression that occurs via horizontal gene transfer can regulate a broad range of specific target genes, resulting in alterations of the phenotype and physiology of host cells.

Ferredoxin-NADP+ reductase from Pseudomonas putida functions as a ferric reductase.

Pseudomonas putida harbors two ferredoxin-NADP(+) reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k(cat)/K(m) value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.

Molecular characterization of fprB (ferredoxin-NADP+ reductase) in Pseudomonas putida KT2440.

The fpr gene, which encodes a ferredoxin-NADP+ reductase, is known to participate in the reversible redox reactions between NADP+/NADPH and electron carriers, such as ferredoxin or flavodoxin. The role of Fpr and its regulatory protein, FinR, in Pseudomonas putida KT2440 on the oxidative and osmotic stress responses has already been characterized [Lee at al. (2006). Biochem. Biophys. Res. Commun. 339, 1246-1254]. In the genome of P. putida KT2440, another Fpr homolog (FprB) has a 35.3% amino acid identity with Fpr. The fprB gene was cloned and expressed in Escherichia coli. The diaphorase activity assay was conducted using purified FprB to identify the function of FprB. In contrast to the fpr gene, the induction of fprB was not affected by oxidative stress agents, such as paraquat, menadione, H2O2 and t-butyl hydroperoxide. However, a higher level of fprB induction was observed under osmotic stress. Targeted disruption of fprB by homologous recombination resulted in a growth defect under high osmotic conditions. Recovery of oxidatively damaged aconitase activity was faster for the fprB mutant than for the fpr mutant, yet still slower than that for the wild type. Therefore, these data suggest that the catalytic function of FprB may have evolved to augment the function of Fpr in P. putida KT2440.