Escherichia coli possessing the dihydroxyacetone phosphate shunt utilize 5'-deoxynucleosides for growth.

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine, 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in Escherichia coli ATCC 25922, when introduced into E. coli K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic E. coli lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt. IMPORTANCE: The acquisition and utilization of organic compounds that serve as growth substrates are essential for Escherichia coli to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5'-deoxy-nucleoside byproducts, 5'-methylthioadenosine and 5'-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic E. coli, but its function in E. coli possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in E. coli strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by E. coli with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments.

Everything but the Kitchen Sink: An Analysis of Bacterial and Chemical Contaminants Found in Syringe Residue From People Who Inject Drugs.

Background: People who inject drugs (PWID) are at high risk of severe wounds, invasive infections, and overdoses. To date, there are few data on the bacterial and chemical contaminants PWID are exposed to when using illicitly manufactured fentanyls and stimulants. Methods: Previously used injection drug use equipment was recovered in St Louis, Missouri, by harm reduction organizations over a 12-month period. Syringe residue was analyzed for bacterial contaminants by routine culturing followed by whole genome sequencing of single bacterial isolates. Chemical adulterants in syringe residue were identified by liquid chromatography-mass spectrometry. Results: Bacteria were cultured from 58.75% of 160 syringes analyzed. Polymicrobial growth was common and was observed in 23.75% of samples. Bacillus cereus was the most common pathogen present and was observed in 20.6% of syringe residues, followed closely by Staphylococcus aureus at 18.8%. One hundred syringes underwent mass spectrometry, which demonstrated that chemical adulterants were common and included caffeine, diphenhydramine, lidocaine, quinine, and xylazine. Conclusions: Analysis of syringe residue from discarded drug use equipment demonstrates both chemical and biological contaminants, including medically important pathogens and adulterants.

Yersiniabactin is a quorum-sensing autoinducer and siderophore in uropathogenic Escherichia coli.

Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

Utilization of 5'-deoxy-nucleosides as Growth Substrates by Extraintestinal Pathogenic E. coli via the Dihydroxyacetone Phosphate Shunt.

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for methylation of biological molecules, synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine (SAH), 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). One of the most prevalent pathways found in bacteria for the metabolism of MTA and 5dAdo is the DHAP shunt, which converts these compounds into dihydroxyacetone phosphate (DHAP) and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Here we show that in Extraintestinal Pathogenic E. coil (ExPEC), the DHAP shunt serves none of these roles in any significant capacity, but rather physiologically functions as an assimilation pathway for use of MTA and 5dAdo as growth substrates. This is further supported by the observation that when MTA is the substrate for the ExPEC DHAP shunt, the sulfur components is not significantly recycled back to methionine, but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. While the pathway is active both aerobically and anaerobically, it only supports aerobic ExPEC growth, suggesting that it primarily functions in oxygenic extraintestinal environments like blood and urine versus the predominantly anoxic gut. This reveals a heretofore overlooked role of the DHAP shunt in carbon assimilation and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt.

E. coli catheter-associated urinary tract infections are associated with distinctive virulence and biofilm gene determinants.

Urinary catheterization facilitates urinary tract colonization by E. coli and increases infection risk. Here, we aimed to identify strain-specific characteristics associated with the transition from colonization to infection in catheterized patients. In a single-site study population, we compared E. coli isolates from patients with catheter-associated asymptomatic bacteriuria (CAASB) to those with catheter-associated urinary tract infection (CAUTI). CAUTI isolates were dominated by a phylotype B2 subclade containing the multidrug-resistant ST131 lineage relative to CAASB isolates, which were phylogenetically more diverse. A distinctive combination of virulence-associated genes was present in the CAUTI-associated B2 subclade. Catheter-associated biofilm formation was widespread among isolates and did not distinguish CAUTI from CAASB strains. Preincubation with CAASB strains could inhibit catheter colonization by multiple ST131 CAUTI isolates. Comparative genomic analysis identified a group of variable genes associated with high catheter biofilm formation present in both CAUTI and CAASB strains. Among these, ferric citrate transport (Fec) system genes were experimentally associated with enhanced catheter biofilm formation using reporter and fecA deletion strains. These results are consistent with a variable role for catheter biofilm formation in promoting CAUTI by ST131-like strains or resisting CAUTI by lower-risk strains that engage in niche exclusion.

Outcomes of Partial Oral Antibiotic Treatment for Complicated Staphylococcus aureus Bacteremia in People Who Inject Drugs.

BACKGROUND: Staphylococcus aureus represents the leading cause of complicated bloodstream infections among persons who inject drugs (PWID). Standard of care (SOC) intravenous (IV) antibiotics result in high rates of treatment success but are not feasible for some PWID. Transition to oral antibiotics may represent an alternative treatment option. METHODS: We evaluated all adult patients with a history of injection drug use hospitalized from January 2016 through December 2021 with complicated S. aureus bloodstream infections, including infective endocarditis, epidural abscess, vertebral osteomyelitis, and septic arthritis. Patients were compared by antibiotic treatment (standard of care intravenous [SOC IV] antibiotics, incomplete IV therapy, or transition from initial IV to partial oral) using the primary composite endpoint of death or readmission from microbiologic failure within 90 days of discharge. RESULTS: Patients who received oral antibiotics after an incomplete IV antibiotic course were significantly less likely to experience microbiologic failure or death than patients discharged without oral antibiotics (P < .001). There was no significant difference in microbiologic failure rates when comparing patients who were discharged on partial oral antibiotics after receiving at least 10 days of IV antibiotics with SOC regimens (P > .9). CONCLUSIONS: Discharge of PWID with partially treated complicated S. aureus bacteremias without oral antibiotics results in high rates of morbidity and should be avoided. For PWID hospitalized with complicated S. aureus bacteremias who have received at least 10 days of effective IV antibiotic therapy after clearance of bacteremia, transition to oral antibiotics with outpatient support represents a potential alternative if the patient does not desire SOC IV antibiotic therapy.

Staphylococcus aureus injection drug use-associated bloodstream infections are propagated by community outbreaks of diverse lineages.

Background: The ongoing injection drug use (IDU) crisis in the United States has been complicated by an emerging epidemic of Staphylococcus aureus IDU-associated bloodstream infections (IDU-BSI). Methods: We performed a case-control study comparing S. aureus IDU-BSI and non-IDU BSI cases identified in a large US Midwestern academic medical center between Jan 1, 2016 and Dec 21, 2019. We obtained the whole-genome sequences of 154 S. aureus IDU-BSI and 91 S. aureus non-IDU BSI cases, which were matched with clinical data. We performed phylogenetic and comparative genomic analyses to investigate clonal expansion of lineages and molecular features characteristic of IDU-BSI isolates. Results: Here we show that patients with IDU-BSI experience longer durations of bacteremia and have lower medical therapy completion rates. In phylogenetic analyses, 45/154 and 1/91 contemporaneous IDU-BSI and non-IDU BSI staphylococcal isolates, respectively, group into multiple, unique clonal clusters, revealing that pathogen community transmission distinctively spurs IDU-BSI. Lastly, multiple S. aureus lineages deficient in canonical virulence genes are overrepresented among IDU-BSI, which may contribute to the distinguishable clinical presentation of IDU-BSI cases. Conclusions: We identify clonal expansion of multiple S. aureus lineages among IDU-BSI isolates, but not non-IDU BSI isolates, in a community with limited access to needle exchange facilities. In the setting of expanding numbers of staphylococcal IDU-BSI cases consideration should be given to treating IDU-associated invasive staphylococcal infections as a communicable disease.

A nitrogenase-like enzyme system catalyzes methionine, ethylene, and methane biogenesis.

Bacterial production of gaseous hydrocarbons such as ethylene and methane affects soil environments and atmospheric climate. We demonstrate that biogenic methane and ethylene from terrestrial and freshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway. This pathway, present in numerous species, uses a nitrogenase-like reductase that is distinct from known nitrogenases and nitrogenase-like reductases and specifically functions in C-S bond breakage to reduce ubiquitous and appreciable volatile organic sulfur compounds such as dimethyl sulfide and (2-methylthio)ethanol. Liberated methanethiol serves as the immediate precursor to methionine, while ethylene or methane is released into the environment. Anaerobic ethylene production by this pathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depleted soils. Methane production reveals an additional bacterial pathway distinct from archaeal methanogenesis.

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli.

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO2 via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

Two Distinct Aerobic Methionine Salvage Pathways Generate Volatile Methanethiol in Rhodopseudomonas palustris.

5'-Methyl-thioadenosine (MTA) is a dead-end, sulfur-containing metabolite and cellular inhibitor that arises from S-adenosyl-l-methionine-dependent reactions. Recent studies have indicated that there are diverse bacterial methionine salvage pathways (MSPs) for MTA detoxification and sulfur salvage. Here, via a combination of gene deletions and directed metabolite detection studies, we report that under aerobic conditions the facultatively anaerobic bacterium Rhodopseudomonas palustris employs both an MTA-isoprenoid shunt identical to that previously described in Rhodospirillum rubrum and a second novel MSP, both of which generate a methanethiol intermediate. The additional R. palustris aerobic MSP, a dihydroxyacetone phosphate (DHAP)-methanethiol shunt, initially converts MTA to 2-(methylthio)ethanol and DHAP. This is identical to the initial steps of the recently reported anaerobic ethylene-forming MSP, the DHAP-ethylene shunt. The aerobic DHAP-methanethiol shunt then further metabolizes 2-(methylthio)ethanol to methanethiol, which can be directly utilized by O-acetyl-l-homoserine sulfhydrylase to regenerate methionine. This is in contrast to the anaerobic DHAP-ethylene shunt, which metabolizes 2-(methylthio)ethanol to ethylene and an unknown organo-sulfur intermediate, revealing functional diversity in MSPs utilizing a 2-(methylthio)ethanol intermediate. When MTA was fed to aerobically growing cells, the rate of volatile methanethiol release was constant irrespective of the presence of sulfate, suggesting a general housekeeping function for these MSPs up through the methanethiol production step. Methanethiol and dimethyl sulfide (DMS), two of the most important compounds of the global sulfur cycle, appear to arise not only from marine ecosystems but from terrestrial ones as well. These results reveal a possible route by which methanethiol might be biologically produced in soil and freshwater environments.IMPORTANCE Biologically available sulfur is often limiting in the environment. Therefore, many organisms have developed methionine salvage pathways (MSPs) to recycle sulfur-containing by-products back into the amino acid methionine. The metabolically versatile bacterium Rhodopseudomonas palustris is unusual in that it possesses two RuBisCOs and two RuBisCO-like proteins. While RuBisCO primarily serves as the carbon fixation enzyme of the Calvin cycle, RuBisCOs and certain RuBisCO-like proteins have also been shown to function in methionine salvage. This work establishes that only one of the R. palustris RuBisCO-like proteins functions as part of an MSP. Moreover, in the presence of oxygen, to salvage sulfur, R. palustris employs two pathways, both of which result in production of volatile methanethiol, a key compound of the global sulfur cycle. When total available sulfur was plentiful, methanethiol was readily released into the environment. However, when sulfur became limiting, methanethiol release decreased, presumably due to methanethiol utilization to regenerate needed methionine.

Microbial pathway for anaerobic 5'-methylthioadenosine metabolism coupled to ethylene formation.

Numerous cellular processes involving S-adenosyl-l-methionine result in the formation of the toxic by-product, 5'-methylthioadenosine (MTA). To prevent inhibitory MTA accumulation and retain biologically available sulfur, most organisms possess the "universal" methionine salvage pathway (MSP). However, the universal MSP is inherently aerobic due to a requirement of molecular oxygen for one of the key enzymes. Here, we report the presence of an exclusively anaerobic MSP that couples MTA metabolism to ethylene formation in the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris In vivo metabolite analysis of gene deletion strains demonstrated that this anaerobic MSP functions via sequential action of MTA phosphorylase (MtnP), 5-(methylthio)ribose-1-phosphate isomerase (MtnA), and an annotated class II aldolase-like protein (Ald2) to form 2-(methylthio)acetaldehyde as an intermediate. 2-(Methylthio)acetaldehyde is reduced to 2-(methylthio)ethanol, which is further metabolized as a usable organic sulfur source, generating stoichiometric amounts of ethylene in the process. Ethylene induction experiments using 2-(methylthio)ethanol versus sulfate as sulfur sources further indicate anaerobic ethylene production from 2-(methylthio)ethanol requires protein synthesis and that this process is regulated. Finally, phylogenetic analysis reveals that the genes corresponding to these enzymes, and presumably the pathway, are widespread among anaerobic and facultatively anaerobic bacteria from soil and freshwater environments. These results not only establish the existence of a functional, exclusively anaerobic MSP, but they also suggest a possible route by which ethylene is produced by microbes in anoxic environments.

Metabolic Regulation as a Consequence of Anaerobic 5-Methylthioadenosine Recycling in Rhodospirillum rubrum.

UNLABELLED: Rhodospirillum rubrum possesses a novel oxygen-independent, aerobic methionine salvage pathway (MSP) for recycling methionine from 5-methylthioadenosine (MTA), the MTA-isoprenoid shunt. This organism can also metabolize MTA as a sulfur source under anaerobic conditions, suggesting that the MTA-isoprenoid shunt may also function anaerobically as well. In this study, deep proteomics profiling, directed metabolite analysis, and reverse transcriptase quantitative PCR (RT-qPCR) revealed metabolic changes in response to anaerobic growth on MTA versus sulfate as sole sulfur source. The abundance of protein levels associated with methionine transport, cell motility, and chemotaxis increased in the presence of MTA over that in the presence of sulfate. Purine salvage from MTA resulted primarily in hypoxanthine accumulation and a decrease in protein levels involved in GMP-to-AMP conversion to balance purine pools. Acyl coenzyme A (acyl-CoA) metabolic protein levels for lipid metabolism were lower in abundance, whereas poly-ß-hydroxybutyrate synthesis and storage were increased nearly 10-fold. The known R. rubrum aerobic MSP was also shown to be upregulated, to function anaerobically, and to recycle MTA. This suggested that other organisms with gene homologues for the MTA-isoprenoid shunt may also possess a functioning anaerobic MSP. In support of our previous findings that ribulose-1,5-carboxylase/oxygenase (RubisCO) is required for an apparently purely anaerobic MSP, RubisCO transcript and protein levels both increased in abundance by over 10-fold in cells grown anaerobically on MTA over those in cells grown on sulfate, resulting in increased intracellular RubisCO activity. These results reveal for the first time global metabolic responses as a consequence of anaerobic MTA metabolism compared to using sulfate as the sulfur source. IMPORTANCE: In nearly all organisms, sulfur-containing byproducts result from many metabolic reactions. Unless these compounds are further metabolized, valuable organic sulfur is lost and can become limiting. To regenerate the sulfur-containing amino acid methionine, organisms typically employ one of several variations of a "universal" methionine salvage pathway (MSP). A common aspect of the universal MSP is a final oxygenation step. This work establishes that the metabolically versatile bacterium Rhodospirillum rubrum employs a novel MSP that does not require oxygen under either aerobic or anaerobic conditions. There is also a separate, dedicated anaerobic MTA metabolic route in R. rubrum This work reveals global changes in cellular metabolism in response to anaerobic MTA metabolism compared to using sulfate as a sulfur source. We found that cell mobility and transport were enhanced, along with lipid, nucleotide, and carbohydrate metabolism, when cells were grown in the presence of MTA.