Title: Traumatic Brain Injury Common Among Homeless Men
Category: Health News
Created: 4/30/2014 12:35:00 PM
Last Editorial Review: 5/1/2014 12:00:00 AM

Title: Depression, Weapons May Be More Common for Bullied Teens
Category: Health News
Created: 4/27/2015 12:00:00 AM
Last Editorial Review: 4/27/2015 12:00:00 AM

Title: Study Casts Doubt on Use of Common Antibiotic for UTIs in Women
Category: Health News
Created: 4/30/2015 12:00:00 AM
Last Editorial Review: 5/1/2015 12:00:00 AM

Title: Heroin Use Surges Among Whites Who Abuse Prescription Painkillers
Category: Health News
Created: 4/30/2015 12:00:00 AM
Last Editorial Review: 5/1/2015 12:00:00 AM

Title: Rates of Severe Obesity Among U.S. Kids Still Rising: Study
Category: Health News
Created: 4/26/2016 12:00:00 AM
Last Editorial Review: 4/26/2016 12:00:00 AM

Title: Antibody Shot Protects Monkeys From HIV-Like Infection
Category: Health News
Created: 4/27/2016 12:00:00 AM
Last Editorial Review: 4/28/2016 12:00:00 AM

Title: Seniors Often Have Trouble Managing Money, Medicines
Category: Health News
Created: 4/28/2017 12:00:00 AM
Last Editorial Review: 5/1/2017 12:00:00 AM

Title: Low-Dose Aspirin May Lower Risk for Common Breast Cancer by 20 Percent
Category: Health News
Created: 5/1/2017 12:00:00 AM
Last Editorial Review: 5/2/2017 12:00:00 AM

Title: Mom's Money Worries May Mean Smaller Baby
Category: Health News
Created: 5/3/2017 12:00:00 AM
Last Editorial Review: 5/4/2017 12:00:00 AM

Title: After Age 50, Yearly Eye Checks May Catch Common Cause of Vision Loss
Category: Health News
Created: 4/28/2018 12:00:00 AM
Last Editorial Review: 4/30/2018 12:00:00 AM

Title: End-of-Life Care Saves Money
Category: Health News
Created: 4/30/2018 12:00:00 AM
Last Editorial Review: 5/1/2018 12:00:00 AM

Title: Study Supports Radiation for Early, Hormone-Driven Breast Cancer
Category: Health News
Created: 4/26/2019 12:00:00 AM
Last Editorial Review: 4/29/2019 12:00:00 AM

Title: Autism Diagnoses Reliable at 14 Months, Study Finds
Category: Health News
Created: 4/29/2019 12:00:00 AM
Last Editorial Review: 4/30/2019 12:00:00 AM

Title: Treatments Targeting Social Behavior Hormone Show Promise With Autism
Category: Health News
Created: 5/1/2019 12:00:00 AM
Last Editorial Review: 5/2/2019 12:00:00 AM

Title: Male-Hormone Gene May Help Cause Polycystic Ovary Syndrome
Category: Health News
Created: 5/1/2019 12:00:00 AM
Last Editorial Review: 5/2/2019 12:00:00 AM

Title: Overdose Attempts Skyrocket Among Teens, Young Adults: Study
Category: Health News
Created: 5/1/2019 12:00:00 AM
Last Editorial Review: 5/2/2019 12:00:00 AM

Title: More Money, Better Heart Health? Not Always
Category: Health News
Created: 4/28/2020 12:00:00 AM
Last Editorial Review: 4/28/2020 12:00:00 AM

Diamond-Blackfan anemia: A genetic condition affecting the bone marrow that leads to anemia (low numbers of red blood cells) and often, birth defects. It affects five to 10 out of every million babies born in the U.S. A number of genetic mutations have been identified that can cause Diamond-Blackfan anemia, and it is inherited in an autosomal dominant pattern, meaning that only one copy of the defective gene is needed to cause the disorder. In about 45% of cases, those affected inherit a defective gene from one parent. In the remaining cases, a new mutation (change in the gene) occurs in people who do not have the condition in their family.

Symptoms and signs of Diamond-Blackfan anemia usually are seen during the first year of life and relate to the anemia and shortage of oxygen delivery to tissues from the low numbers of red blood cells. These symptoms include pallor, fatigue, and weakness. People with this disorder are at increased risk for developing myelodysplastic syndrome, another bone marrow disorder, acute myeloid leukemia (AML), and a type of bone cancer known as osteosarcoma. The birth defects that can accompany the bone marrow disorders are seen in about half of those affected and can vary in severity. Associated birth defects can include small head size (microcephaly); wide-set eyes (hypertelorism); droopy eyelids (ptosis); a low frontal hairline; a broad, flat bridge of the nose; small, low-set ears; and small lower jaw (micrognathia). Cleft palate and/or cleft lip may also be present. Slow growth, eye problems, and kidney problems are sometimes associated with the condition. Red blood cell transfusions and corticosteroid medications are the main treatments for Diamond-Blackfan anemia. Stem cell transplants have been performed successfully in some children with the condition.

Title: Thousands of COVID-19 Cases Among Inmates and Staff at U.S. Prisons and Jails: CDC
Category: Health News
Created: 5/7/2020 12:00:00 AM
Last Editorial Review: 5/8/2020 12:00:00 AM

Title: Bacteria May Be a Player in Diabetes Among Very Obese
Category: Health News
Created: 3/12/2020 12:00:00 AM
Last Editorial Review: 3/13/2020 12:00:00 AM

Title: Heart Attacks, Strokes Are Declining Among People With Diabetes
Category: Health News
Created: 5/1/2020 12:00:00 AM
Last Editorial Review: 5/4/2020 12:00:00 AM

Title: Hantavirus Pulmonary Syndrome (HPS)
Category: Diseases and Conditions
Created: 4/21/2010 12:00:00 AM
Last Editorial Review: 3/24/2020 12:00:00 AM

Title: Pneumonia More Deadly Than Hip Fractures for Hospitalized Seniors
Category: Health News
Created: 4/23/2020 12:00:00 AM
Last Editorial Review: 4/24/2020 12:00:00 AM

Title: Money Not a Good Measure of Your Self-Worth
Category: Health News
Created: 4/14/2020 12:00:00 AM
Last Editorial Review: 4/15/2020 12:00:00 AM

Title: How Common Are STDs? Most People Have No Clue
Category: Health News
Created: 2/25/2020 12:00:00 AM
Last Editorial Review: 2/25/2020 12:00:00 AM

Title: New Study Shakes Up Thinking on Hormone Replacement Therapy
Category: Health News
Created: 12/13/2019 12:00:00 AM
Last Editorial Review: 12/13/2019 12:00:00 AM

Title: Pot Use Among U.S. Seniors Nearly Doubled in 3 Years
Category: Health News
Created: 2/24/2020 12:00:00 AM
Last Editorial Review: 2/25/2020 12:00:00 AM

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from ¹³C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

ABSTRACT

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11b^hi Ly6C^hi inflammatory monocytes and followed by the establishment of a CD11b^hi Ly6C^lo CX₃CR1⁺ macrophage population in the muscle upon recovery. Selective modulation of CD11b^hi Ly6C^hi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX₃CR1⁺ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.

IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX₃CR1⁺ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX₃CR1⁺ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

ABSTRACT

Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K. pneumoniae, Escherichia coli, and Salmonella. TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa. In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniae. tmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.

IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae. Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

ABSTRACT

Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.

IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

ABSTRACT

One of the primary functions of the mucosal barrier, found lining epithelial cells, is to serve as a first-line of defense against microbial pathogens. The major structural components of mucus are heavily glycosylated proteins called mucins. Mucins are key components of the innate immune system as they aid in the clearance of pathogens and can decrease pathogen virulence. It has also been recently reported that individual mucins and derived glycans can attenuate the virulence of the human pathogen Pseudomonas aeruginosa. Here, we show data indicating that mucins not only play a role in host defense but that they can also be subverted by P. aeruginosa to cause disease. We found that the mucin MUL-1 and mucin-derived monosaccharides N-acetyl-galactosamine and N-acetylglucosamine are required for P. aeruginosa killing of Caenorhabditis elegans. We also found that the defective adhesion of P. aeruginosa to human lung alveolar epithelial cells, deficient in the mucin MUC1, can be reversed by the addition of individual monosaccharides. The monosaccharides identified in this study are found in a wide range of organisms where they act as host factors required for bacterial pathogenesis. While mucins in C. elegans lack sialic acid caps, which makes their monosaccharides readily available, they are capped in other species. Pathogens such as P. aeruginosa that lack sialidases may rely on enzymes from other bacteria to utilize mucin-derived monosaccharides.

IMPORTANCE One of the first lines of defense present at mucosal epithelial tissues is mucus, which is a highly viscous material formed by mucin glycoproteins. Mucins serve various functions, but importantly they aid in the clearance of pathogens and debris from epithelial barriers and serve as innate immune factors. In this study, we describe a requirement of host monosaccharides, likely derived from host mucins, for the ability of Pseudomonas aeruginosa to colonize the intestine and ultimately cause death in Caenorhabditis elegans. We also demonstrate that monosaccharides alter the ability of bacteria to bind to both Caenorhabditis elegans intestinal cells and human lung alveolar epithelial cells, suggesting that there are conserved mechanisms underlying host-pathogen interactions in a range of organisms. By gaining a better understanding of pathogen-mucin interactions, we can develop better approaches to protect against pathogen infection.

ABSTRACT

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa. We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD⁺ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.

IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa. This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach.

ABSTRACT

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin–laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.

IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner.

ABSTRACT

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.

IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

ABSTRACT

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.

IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains.

ABSTRACT

The recent discovery of complete ammonia oxidizers (comammox) contradicts the paradigm that chemolithoautotrophic nitrification is always catalyzed by two different microorganisms. However, our knowledge of the survival strategies of comammox in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Analyses of genomes and in situ transcriptomes of four comammox organisms from two full-scale WWTPs revealed that comammox were active and showed a surprisingly high metabolic versatility. A gene cluster for the utilization of urea and a gene encoding cyanase suggest that comammox may use diverse organic nitrogen compounds in addition to free ammonia as the substrates. The comammox organisms also encoded the genomic potential for multiple alternative energy metabolisms, including respiration with hydrogen, formate, and sulfite as electron donors. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely help comammox survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. One of the comammox strains acquired from the anaerobic tank encoded and transcribed genes involved in homoacetate fermentation or in the utilization of exogenous acetate, both pathways being unexpected in a nitrifying bacterium. Surprisingly, this strain also encoded a respiratory nitrate reductase which has not yet been found in any other Nitrospira genome and might confer a selective advantage to this strain over other Nitrospira strains in anoxic conditions.

IMPORTANCE The discovery of comammox in the genus Nitrospira changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and—among nitrifiers—unsurpassed ecophysiological versatility could make comammox Nitrospira an interesting target for optimizing nitrification in current and future bioreactor configurations.

ABSTRACT

The global stress response controlled by the alternative sigma factor RpoS protects enteric bacteria from a variety of environmental stressors. The role of RpoS in other, nonenteric bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, is less well understood. Here, we employed experimental social evolution to reveal that cooperative behavior via secreted public goods is an important function in the RpoS response of P. aeruginosa. Using whole-genome sequencing, we identified rpoS loss-of-function mutants among isolates evolved in a protein growth medium that requires extracellular proteolysis. We found that rpoS mutants comprise up to 25% of the evolved population and that they behave as social cheaters, with low fitness in isolation but high fitness in mixed culture with the cooperating wild type. We conclude that rpoS mutants cheat because they exploit an RpoS-controlled public good produced by the wild type, the secreted aminopeptidase PaAP, and because they do not carry the metabolic costs of expressing PaAP and many other gene products in the large RpoS regulon. Our results suggest that PaAP is an integral part of a proteolytic sequence in P. aeruginosa that permits the utilization of protein as a nutrient source. Our work broadens the scope of stress response functions in bacteria.

IMPORTANCE Bacterial stress responses are generally considered protective measures taken by individual cells. Enabled by an experimental evolution approach, we describe a contrasting property, collective nutrient acquisition, in the RpoS-dependent stress response of the opportunistic human pathogen P. aeruginosa. Specifically, we identify the secreted P. aeruginosa aminopeptidase (PaAP) as an essential RpoS-controlled function in extracellular proteolysis. As a secreted "public good," PaAP permits cheating by rpoS mutants that save the metabolic costs of expressing RpoS-controlled genes dispensable under the given growth conditions. Proteolytic enzymes are important virulence factors in P. aeruginosa pathogenesis and constitute a potential target for antimicrobial therapy. More broadly, our work contributes to recent findings in higher organisms that stress affects not only individual fitness and competitiveness but also cooperative behavior.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized "EDEMP cycle" (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each.

IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer ("fluxomic") analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed ("fluxed") through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this "blueprint" is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

ABSTRACT

Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action.

IMPORTANCE Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.

ABSTRACT

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca²⁺ channel Ca_V3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As Ca_V3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis. The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.

IMPORTANCE Bordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca²⁺ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

ABSTRACT

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated adcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the adcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated adcAII strains. However, transformation of adcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated adcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of adcAII. Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.

IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the adcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and adcAII, further investigation of which could further characterize mechanisms of capsule regulation.

ABSTRACT

Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa. Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducens fabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction.

IMPORTANCE Geobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria.

ABSTRACT

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.

IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

ABSTRACT

Obesity and associated metabolic disorders are worldwide public health issues. The gut microbiota plays a key role in the pathophysiology of diet-induced obesity. Glycerol monolaurate (GML) is a widely consumed food emulsifier with antibacterial properties. Here, we explore the anti-obesity effect of GML (1,600 mg/kg of body weight) in high-fat diet (HFD)-fed mice. HFD-fed mice were treated with 1,600 mg/kg GML. Integrated microbiome, metabolome, and transcriptome analyses were used to systematically investigate the metabolic effects of GML, and antibiotic treatment was used to assess the effects of GML on the gut microbiota. Our data indicated that GML significantly reduced body weight and visceral fat deposition, improved hyperlipidemia and hepatic lipid metabolism, and ameliorated glucose homeostasis and inflammation in HFD-fed mice. Importantly, GML modulated HFD-induced gut microbiota dysbiosis and selectively increased the abundance of Bifidobacterium pseudolongum. Antibiotic treatment abolished all the GML-mediated metabolic improvements. A multiomics (microbiome, metabolome, and transcriptome) association study showed that GML significantly modulated glycerophospholipid metabolism, and the abundance of Bifidobacterium pseudolongum strongly correlated with the metabolites and genes that participated in glycerophospholipid metabolism. Our results indicated that GML may be provided for obesity prevention by targeting the gut microbiota and regulating glycerophospholipid metabolism.

ABSTRACT

In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like β-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 Klebsiella isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for β-lactam permeation. Sequence similarity network analysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic Klebsiella pneumoniae strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since K. pneumoniae is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in K. pneumoniae.

IMPORTANCE Klebsiella pneumoniae is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant K. pneumoniae (CRKP). The outer membrane of K. pneumoniae forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of K. pneumoniae, demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and β-lactamase inhibitors could be effective on porin-deficient K. pneumoniae. Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of K. pneumoniae and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for K. pneumoniae infections, given the way porin expression will be modulated to confer phage-resistant—and collateral drug-resistant—phenotypes in K. pneumoniae.

ABSTRACT

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca²⁺-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol—and concomitant with the proteomic changes—lanthanum (La³⁺) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy.

IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La³⁺) is mostly substrate specific and that La³⁺ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

The nitrate transport accessory protein OsNAR2 plays a critical role in root-growth responses to nitrate and nitrate acquisition in rice (Oryza sativa). In this study, a pull-down assay combined with yeast two-hybrid and coimmunoprecipitation analyses revealed that OsNAR2.1 interacts with OsNIT1 and OsNIT2. Moreover, an in vitro nitrilase activity assay indicated that indole-3-acetonitrile (IAN) is hydrolyzed to indole-3-acetic acid (IAA) by OsNIT1, the activity of which was enhanced 3- to 4-fold by OsNIT2 and in excess of 5- to 8-fold by OsNAR2.1. Knockout (KO) of OsNAR2.1 was accompanied by repressed expression of both OsNIT1 and OsNIT2, whereas KO of OsNIT1 and OsNIT2 in the osnit1 and osnit2 mutant lines did not affect expression of OsNAR2.1 or the root nitrate acquisition rate. osnit1 and osnit2 displayed decreased primary root length and lateral root density. Double KO of OsNAR2.1 and OsNIT2 caused further decreases in lateral root density under nitrate supply. Ammonium supply repressed OsNAR2.1 expression whereas it upregulated OsNIT1 and OsNIT2 expression. Both osnit1 and osnit2 showed root growth hypersensitivity to external ammonium; however, less root growth sensitivity to external IAN, higher expression of three IAA-amido synthetase genes, and a lower rate of ³H-IAA movement toward the roots were observed. Taken together, we conclude that the interaction of OsNIT1 and OsNIT2 activated by OsNAR2.1 and nitrogen supply is essential for maintaining root growth possibly via altering the IAA ratio of free to conjugate forms and facilitating its transportation.

The corrinoid B₁₂ is synthesized only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B₁₂ dependence on multiple occasions, and we previously demonstrated that experimental evolution of the non–B₁₂-requiring alga Chlamydomonas reinhardtii in media supplemented with B₁₂ generated a B₁₂-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B₁₂ auxotroph. Our analyses demonstrate that B₁₂ deprivation of metE7 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B₁₂ deprivation also caused a substantial increase in reactive oxygen species, which preceded rapid cell death. Survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B₁₂ limitation and an increase in B₁₂ use efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B₁₂-producing bacterium. Therefore, although an early B₁₂-dependent alga would likely be poorly adapted to coping with B₁₂ deprivation, association with B₁₂-producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B₁₂ limitation better.