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Shin, J.-H.

Publications and source records attributed to Shin, J.-H..

16 recordsLinked to original sources

RELAX does not reproduce its own estimates at default settings, and its output does not show it

Selection-intensity estimates from RELAX are reported as a point value of K with a likelihood-ratio P. We report that, at default settings and on data of ordinary size, the program does not reproduce its own fits. Of 27 enzyme entries refitted under two optimiser configurations, none reproduced its log-likelihood to within 0.01 units; the median change was 103 units, the largest over 3,400, and four verdicts reversed. Eighty null orthologues reproduced none. A byte-identical command returned a distinct likelihood on every repetition, single-threaded, across three releases, and on alignments simulated under the fitted model, where 3.3 per cent of replicates reproduced. The documented random-number seed never reaches the generator when assigned on the command line, yet reads back as the value supplied. PAML localises the cause: its two-ratio model, without site classes, reproduced its log-likelihood for all 288 genes; its site-class models agreed for 27 to 67 per cent. The instability follows the mixture over sites, not the program. The output does not show it: 46 of 410 fits ended with a negative likelihood-ratio statistic, impossible under convergence, and 123 of 410 report a K re-estimated under a domain restriction rather than the unconstrained maximum. Of 234 published studies using RELAX, none reported a seed. Seeding while holding the thread count at one reproduced sixty of sixty runs on twenty genes under two releases; the seed alone reproduced none of five, and no documentation states the second condition. We recommend that fits be repeated and their dispersion published.

evolutionary biology↗

Relaxed selection and convergent loss erode the retained plant-cell-wall-degrading enzymes of ectomycorrhizal fungi

The contraction of the plant-cell-wall-degrading enzyme (PCWDE) repertoire in ectomycorrhizal (ECM) fungi is among the most reproducible results in fungal comparative genomics, and it is routinely read as a loss of decay capacity - a statement about the genes that persist, which counts cannot make. We addressed that question in 183 fungal genomes at two levels and reached opposite conclusions. Gene loss is measurable. On a 182-tip species tree, ECM genomes carry 0.367 times the PCWDE complement of free-living decomposers (phylogenetic generalized least squares; 95% CI 0.295-0.457; P = 1.3 x 10^-15; Pagel's {lambda} = 0.92); the ECM coefficient is not absorbed into the phylogenetic covariance term. The selective regime of the copies that remain is not measurable with the standard branch-partitioned test, and this version retracts the relaxed-selection claims we made for GH6, GH7 and GH28 in version 1. Four independent controls support the retraction. (i) Calibration: applying the identical design to single-copy orthologues, which have no reason to experience a change of selective regime specific to ECM lineages, the relaxation test rejected the null for 35.8% of loci at = 0.05 (29 of 81; 43.9% of the 66 returning an interpretable statistic), against a nominal 5%. (ii) Non-convergence: in 46 of 410 fits the likelihood-ratio statistic was negative, an outcome that cannot occur if the optimisation has converged. (iii) Non-reproducibility: across 80 pairs of fits sharing alignment, tree and branch labels, none recovered its own log-likelihood to within 0.01 units, while the nested model fits computed inside the same runs agreed to within 1.99 units. (iv) Resolution: with 81 to 82 null replicates the smallest attainable empirical P value is about 0.012, against a Bonferroni threshold of 0.0125 for the four families tested here - a margin we had not computed, and one that any expansion of the family set removes. No family-level statement about selective regime is supported by these data. We emphasise the symmetry: we do not claim that these genes are maintained either. The pattern of gene loss described by previous work stands, and is strengthened by phylogenetic correction; what does not stand is the inference from that pattern to the functional state of the genes that remain.

evolutionary biology↗

Context-dependent calibration of Evo2 likelihood with bacterial fitness: a quantitative characterization across five E. coli datasets

DNA foundation models are trained to predict the likelihood of natural sequences, but the calibration between such likelihood scores and laboratory fitness or directly measured molecular phenotypes depends strongly on gene context, sequence divergence from wild-type, and selection regime. We apply zero-shot variant scoring with Evo2 7B ({Delta}LLR, the change in pseudo-log-likelihood between mutant and reference windows) to five E. coli datasets and quantify this context-dependent calibration map. Calibration is strong in two settings. In the Firnberg 2014 deep mutational scan of TEM-1 {beta}-lactamase (13,027 nucleotide-level variants; plasmid-borne enzyme under band-pass ampicillin selection), Evo2 {Delta}LLR tracks measured fitness at Spearman {rho} = 0.545 (95% CI 0.532-0.557; SNV {rho} = 0.606, indel {rho} = 0.521). In the Tenaillon 2012 thermal-evolution dataset, type-stratified, window-tuned scoring reaches Insertion AUROC 0.882 (W = 2,048 bp) and Deletion AUROC 0.846 (W = 4,096 bp). Calibration is decisively absent in the same organism: the Ireland 2020 RegSeq promoter MPRA gives{rho} = 0.011 (95% CI 0.003-0.019; n = 64,665), flat even after -10/-35 mechanism stratification, and the Dewachter 2023 chromosomal-essentials scan (fabZ/lpxC/murA) gives{rho} = 0.041 (95% CI 0.025-0.058). The Papkou 2023 folA combinatorial landscape sits between, at{rho} = 0.237, with a sweep that falls monotonically from {rho} = 0.575 at two mutations from wild-type to {rho} = 0.065 at nine. Pooling per-gene and per-divergence correlations, we fit calibration as an explicit function{rho} = f(sequence divergence from WT, variant context): weighted regression gives a negative divergence coefficient and a negative regulatory-context coefficient (both in the predicted direction; R2 = 0.49) -- an explicit, if illustrative, fit rather than a metaphor. We further test -- and find unsupported -- the intuitive explanation for the residual TEM-1 vs. essentials gap: across five genes the chromosomal essentials are more represented than TEM-1 by raw public-database deposition count yet calibrate far worse (calibration does not track deposition count; if anything, inversely), so simple training over-representation does not explain the gap. Deposited variant diversity is a candidate but remains untested. We therefore reframe Evo2 not as a fitness predictor but as a likelihood predictor whose calibration with fitness is context-dependent. The deliverable is not a DMS pre-screen tool but a quantitative lookup table of when, where, and why the likelihood- fitness gap closes (training-rich plasmid CDS under stringent selection) or opens (chromosomal essentials, native promoter regulatory variants). Even within a single organism, plasmid vs. chromosomal context and strong vs. weak selection yield qualitatively different calibration regimes -- the central finding.

bioinformatics↗

Transcriptomic analysis of the human habenula in schizophrenia

Pathophysiology of many neuropsychiatric disorders, including schizophrenia (SCZD), is linked to habenula (Hb) function. While pharmacotherapies and deep brain stimulation targeting the Hb are emerging as promising therapeutic treatments, little is known about the cell type-specific transcriptomic organization of the human Hb or how it is altered in SCZD. Here we define the molecular neuroanatomy of the human Hb and identify transcriptomic changes in individuals with SCZD compared to neurotypical controls. Utilizing Hb-enriched postmortem human brain tissue, we performed single nucleus RNA-sequencing (snRNA-seq; n=7 neurotypical donors) and identified 17 molecularly defined Hb cell types across 16,437 nuclei, including 3 medial and 7 lateral Hb populations, several of which were conserved between rodents and humans. Single molecule fluorescent in situ hybridization (smFISH; n=3 neurotypical donors) validated snRNA-seq Hb cell types and mapped their spatial locations. Bulk RNA-sequencing and cell type deconvolution in Hb-enriched tissue from 35 individuals with SCZD and 33 neurotypical controls yielded 45 SCZD-associated differentially expressed genes (DEGs, FDR < 0.05), with 32 (71%) unique to Hb-enriched tissue. eQTL analysis identified 717 independent SNP-gene pairs (FDR < 0.05), where either the SNP is a SCZD risk variant (16 pairs) or the gene is a SCZD DEG (7 pairs). eQTL and SCZD risk colocalization analysis identified 16 colocalized genes. These results identify topographically organized cell types with distinct molecular signatures in the human Hb and demonstrate unique genetic changes associated with SCZD, thereby providing novel molecular insights into the role of Hb in neuropsychiatric disorders. One Sentence SummaryTranscriptomic analysis of the human habenula and identification of molecular changes associated with schizophrenia risk and illness state.

neuroscience↗

Genetic and transcriptomic analysis of hyphal constriction based on a novel assay method in the rice blast fungus

An ability of fungi to undergo hyphal constriction is important for fungal ecology and diseases. In the rice blast fungus, Magnaporthe oryzae, hyphal constriction is required to traverse host cell junctions through pit fields. However, little is known about genetic underpinnings of hyphal constriction, except the requirement of a mitogen-activated protein kinase, Pmk1. Here we demonstrate that a simple in vitro assay based on nitrocellulose membrane allows investigation of the genetic basis for hyphal constriction. Using the assay, we found that the constriction limit of M. oryzae hypha lies between 0.22 and 0.3 m, and that a histone modification might be involved in hyphal constriction. RNA-seq experiments combining our assay and {Delta}pmk1 showed that hyphal constriction relies on transcriptional changes of genes implicated primarily in membrane and cell wall-related cellular processes in Pmk1-dependent and/or Pmk1-independent manner. Furthermore, our assays with diverse fungal species suggest correlation between hyphal constriction and fungal lifestyles. Our study reveals that hyphal constriction can be induced without host-derived cues and provides molecular and evolutionary insights into a fundamental process to infection of host plant.

microbiology↗

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway

In tissues with mechanical function, the regulation of remodeling and repair processes is often controlled by mechanosensitive mechanisms; damage to the tissue structure is detected by changes in mechanical stress and strain, stimulating matrix synthesis and repair. While this mechanoregulatory feedback process is well recognized in animals and plants, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress and strain within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, compression, and hydrostatic pressure. In all three cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Mechanosensitivity of VxrAB signaling was lost following removal of the VxrAB stimulating endopeptidase ShyA, suggesting that VxrAB may not be directly sensing mechanical forces, but instead relies on other factors including lytic enzymes in the periplasmic space. Our findings suggest that mechanical signals play an important role in regulating cell wall homeostasis in bacteria. Significance StatementBiological materials with mechanical function (bones, muscle, etc.) are often maintained through mechanosensitive mechanisms, in which damage-induced reductions in stiffness stimulate remodeling and repair processes that restore mechanical function. Here we show that a similar process can occur in bacteria. We find that mechanical stresses in the bacterial cell envelope (the primary load-bearing structure in bacteria) regulate signaling of a two-component system involved in cell wall synthesis. These findings suggest that the mechanical stress state within the cell envelope can contribute to cell wall homeostasis. Furthermore, these findings demonstrate the potential to use mechanical stimuli to regulate gene expression in bacteria.

microbiology↗

The effect of treatment of auxin producing bacterial culture supernatant in combination with organic and inorganic fertilizers

Bacteria that secret plant growth promoting substances are good candidate for field application to improve crop yield. Especially, bacteria that excrete auxin can enhance root growth and settlement of crops to help adapting new soil environment when transplanted. In this study, we applied culture supernatant of an auxin producing bacteria Ignatzschineria sp. CG20001. The bacterial culture supernatant (BCS) was applied in combination with chicken manure and liquid chemical fertilizer. The treatment of bacterial culture supernatant enhanced growth of red kohlrabi, Brassica oleraceae var. gongyloides, especially in combination with chicken manure. To understand mechanisms related higher growth, we compared photosynthetic efficiency by measuring chlorophyll fluorescence and light harvesting antenna pigments composition. The performance index PS II was higher in BCS treated plants, and recovered faster after leaf harvest. Photoprotective mechanism was also increased in BCS treated plants, however, and DTT infiltration, non-photochemical quenching (NPQ) reduced to similar level, implying higher xanthophyll cycle dependent NPQ induction mechanism in BCS treated plants. However, in liquid chemical fertilized plants, BCS treated and non-treated plants showed the opposite pattern in the change of NPQ. The chlorophyll a/b ratio was highest in no-fertilized non-treated control, and lowest in chicken manure and BCS combination, implying impact of nutrition availability between the two groups. Through these results, we conclude that the appropriate combination of organic fertilizer and plant growth promoting bacterial metabolite can improve plant growth more than chemical fertilizers. This study also provides physiological understanding for strategic fertilization regime of organic fertilizer and biofertilizer combination.

plant biology↗

CTCF-mediated Genomic Effects of BART Region on Epstein-Barr Virus Chromatin 3D Structure in Gastric Carcinoma Cells

EBV latent infection in gastric carcinoma (GC) cells is characterized by distinct viral gene expression programs. CCCTC-binding factor (CTCF) is a chromatin structural factor that has been involved in coordinated chromatin interactions between multiple loci of Epstein-Barr virus (EBV) genes. Here, we investigate the role of CTCF in regulating EBV gene expression and chromosome conformation in model of EBV-associated gastric carcinoma (EBVaGC). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) against CTCF revealed 16 CTCF binding sites (BS) in EBV genome of EBVaGC, SNU719 cells. Among the CTCF BSs, one site named as BARTp (BamHI A right transcript promoter) CTCF BS is located at upstream of 11.8-kb BART region (EBV genome: 139724-151554) and was not yet defined its biological functions in EBV life cycle. EBV BART encodes a complex miRNA cluster of highly spliced transcripts that is implicated in EBV cancer pathogenesis. This present study investigated the functional role of the CTCF binding site at BARTp (BARTp CTCF BS) in regulating EBV gene transcription and EBV three-dimensional (3D) genome structure as DNA loop maker. Circular chromatin confirmation capture (4C)-seq and chromatin confirmation capture (3C)-semi-quantitative(sq)PCR assays using SNU719 cells revealed that BARTp CTCF BS interacts with CTCF BSs of LMP1/2, Cp/OriP, and Qp in EBV genome. We generated mutations in BARTp CTCF BS (S13) in bacmids with (BART+) or without (BART-) the 11.8-kb BART transcript unit (B(+/-)). ChIP-qPCR assay demonstrated that CTCF binding was ablated from BARTp in EBV B(+/-) S13- genomes (mutant S13), elevated at several other sites such as LMP1, OriP, and Cp in EBV B(-) (BART-) S13- genome, and decreased at the same sites in EBV B(+) S13- genome. Infection assay showed that BARTp CTCF BS mutation reduced infectivity, while BART transcript deletion has no detectable effects. Gene expression tests showed that EBNA1 was highly downregulated in B(+/-) S13- EBVs related to B(+/-) S13+ EBVs (wild-type S13). LMP1 and BZLF1 were more downregulated in B(-) S13- EBV than B(+) S13- EBV. Taken together, these findings suggest that the CTCF binding and BART region contribute to EBV 3D genome structure via a cluster of DNA loops formed by BARTp CTCF BS (S13) and are important for coordinated viral gene expression and EBV infectivity.

microbiology↗

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and structural integrity. PG is synthesized by two different types of synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by autolytic enzymes to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of endopeptidases, EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, a decline in viability, and continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, without effectively promoting cell elongation. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, the Rod system requires endopeptidase activity (but not necessarily direct interaction with EPs) to promote cell expansion and substantial PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency. ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are required only for substantial PG incorporation mediated by the Rod system, while the aPBPs maintain structural integrity during endopeptidase insufficiency. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the co-ordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

microbiology↗

Although host-related factors are important for the formation of gut microbiota, environmental factors cannot be ignored

The gut microbiome is essential to human health. However, little is known about the influence of the environment versus host-related factors (e.g. genetic background, sex, age, and body mass) in the formation of human intestinal microflora. Here, we present evidence in support of the importance of host-related factors in the establishment and maintenance of individual gut assemblages. We collected fecal samples (n = 249) from 44 Korean naval trainees and 39 healthy people living in Korea over eight weeks and sequenced the bacterial 16S rRNA genes. The following hypotheses were tested: 1) microbiome function is linked to its diversity, community structure, and genetic host-related factors, and 2) preexisting host-related factors have a more significant effect on gut microbiome formation and composition than environmental factors. For each individual, the difference between the initial gut microbiota and that after eight weeks was negligible even though the 44 naval trainees lived in the same area and received the same diet, the same amount of exercise, and the same amount of physical stress during the study. This suggests that host-related factors, rather than environmental factors, is a key determinant of individual gut microflora. Moreover, eight weeks of physical training and experiencing the same environmental conditions resulted in an increase in the species Bifidobacterium, Faecalibacterium, and Roseburia in most trainees, suggesting a healthier intestinal environment. IMPORTANCEIn order to understand the role of human gut microbiome, it is important to know how individuals gut microbiota are formed. In this study, we tested the host-related factors versus environmental factors to affect gut microbiome and found that the former have a more association. However, we also found that the controlled environment give an effect on the gut microflora as well. This study provides preliminary evidence that differences in the formation and diversity of gut microbiota within a population could be determined by host-related factors rather than environmental factors.

microbiology↗

Structural basis of peptidoglycan endopeptidase regulation

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., {beta}-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangement in vivo. SignificanceBacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogen Vibrio cholerae. Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.

microbiology↗

A multifaceted cellular damage repair and prevention pathway promotes high level tolerance to β-lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level {beta}-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that {beta}-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the {Delta}vxrAB mutant. Mutating major iron uptake systems drastically increased {Delta}vxrAB tolerance to {beta}-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against {beta}-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganisms ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance, and highlights the complex secondary responses elicited by antibiotics.

microbiology↗

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

biochemistry↗

Human cortical neural stem cells generate regional organizer states in vitro before committing to excitatory neuronal fates

Better understanding the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here we used RNA-sequencing, cell imaging and lineage tracing of mouse and human in vitro NSCs to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs developed from pluripotency in vitro, they first transitioned through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines varied in these early patterning states leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analysis of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.

neuroscience↗

Spheroplast-mediated carbapenem tolerance in Gram-negative pathogens

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied, yet likely widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae is able to tolerate exposure to the typically bactericidal {beta}-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread tolerance is. Here, we have tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella aerogenes, but not Escherichia coli, exhibit moderate to high levels of tolerance to meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered to wild-type morphology and exponential growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

microbiology↗

Genetic determinants of penicillin tolerance in Vibrio cholerae

Many bacteria are resistant to killing (\"tolerant\") by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae. Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well-characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A Penicillin Binding Proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. TnSeq analyses revealed that LPS and cell wall biogenesis genes as well as the sigma E cell envelope stress response were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than Lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for post-antibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

microbiology↗