scholarly journals Indole-3-Glycerol-Phosphate Synthase Is Recognized by a Cold-Inducible Group II Chaperonin in Thermococcus kodakarensis

2012 ◽  
Vol 78 (11) ◽  
pp. 3806-3815 ◽  
Author(s):  
Le Gao ◽  
Atsushi Danno ◽  
Sayaka Fujii ◽  
Wakao Fukuda ◽  
Tadayuki Imanaka ◽  
...  
Keyword(s):  
Cell Growth ◽  
Minimal Medium ◽  
Terminal Region ◽  
Amino Acid Residues ◽  
Glycerol Phosphate ◽  
Content Type ◽  
Thermococcus Kodakarensis ◽  
Mascot Score ◽  
Group Ii Chaperonin ◽  
Phosphate Synthase

ABSTRACTThermococcus kodakarensisoptimally grows at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB. Gene disruptants DA1 (ΔcpkA) and DB1 (ΔcpkB) showed decreased cell growth at 60°C and 93°C, respectively. The DB2 mutant (ΔcpkA∷cpkBΔcpkB), whosecpkBgene was expressed under the control of thecpkApromoter, did not grow at 60°C, and the DB3 mutant [ΔcpkA(1–524)∷cpkB(1–524) ΔcpkB], whose CpkA amino acid residues 1 to 524 were replaced with corresponding CpkB residues that maintained the C-terminal region intact, grew at 60°C, implying that the CpkA C-terminal region plays a key role in cell growth at 60°C. To screen for specific CpkA target proteins, comparative pulldown studies with anti-Cpk were performed using cytoplasmic fractions from DA1 cells cultivated at 93°C and DB1 cells cultivated at 60°C. Among the proteins coprecipitated with anti-Cpk, TK0252, encoding indole-3-glycerol-phosphate synthase (TrpC), showed the highest Mascot score. Counter-pulldown experiments were also performed on DA1 and DB1 extracts using anti-TrpC. CpkA coimmunoprecipitated with anti-TrpC while CpkB did not. The results obtained indicate that TrpC is a specific target for CpkA. The effects of Cpks on denatured TrpC were then examined. The refolding of partially denatured TrpC was accelerated by the addition of CpkA but not by adding CpkB. DA1 cells grew optimally in minimal medium only in the presence of tryptophan but hardly grew in the absence of tryptophan at 60°C. It has been suggested that a lesion of functional TrpC is caused bycpkAdisruption, resulting in tryptophan auxotrophy.

scholarly journals A Mutant Chaperonin That Is Functional at Lower Temperatures Enables Hyperthermophilic Archaea To Grow under Cold-Stress Conditions

2015 ◽  
Vol 197 (16) ◽  
pp. 2642-2652 ◽  
Author(s):  
Le Gao ◽  
Tadayuki Imanaka ◽  
Shinsuke Fujiwara
Keyword(s):  
Atpase Activity ◽  
Cold Stress ◽  
High Temperatures ◽  
Terminal Region ◽  
Stress Conditions ◽  
Hyperthermophilic Archaea ◽  
Gene Copy ◽  
Content Type ◽  
Single Base ◽  
Thermococcus Kodakarensis

ABSTRACTThermococcus kodakarensisgrows optimally at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively. The two chaperonins share a high sequence identity (77%), except in their C-terminal regions. CpkA, which contains tandem repeats of a GGM motif, shows its highest ATPase activity at 60°C to 70°C, whereas CpkB shows its highest activity at temperatures higher than 90°C. To clarify the effects of changes in ATPase activity on chaperonin function at lower temperatures, various CpkA variants were constructed by introducing single point mutations into the C-terminal region. A CpkA variant in which Glu530 was replaced with Gly (CpkA-E530G) showed increased ATPase activity, with its highest activity at 50°C. The efficacy of the CpkA variants against denatured indole-3-glycerol-phosphate synthase ofT. kodakarensis(TrpCTk), which is a CpkA target, was then examinedin vitro. CpkA-E530G was more effective than wild-type CpkA at facilitating the refolding of chemically unfolded TrpCTkat 50°C. The effect ofcpkA-E530Gon cell growth was then examined by introducingcpkA-E530Ginto the genome ofT. kodakarensisKU216 (pyrF). The mutant strain, DA4 (pyrF cpkA-E530G), grew as well as the parental KU216 strain at 60°C. In contrast, DA4 grew more vigorously than KU216 at 50°C. These results suggested that the CpkA-E530G mutation prevented cold denaturation of proteins under cold-stress conditions, thereby enabling cells to grow in cooler environments. Thus, a single base pair substitution in a chaperonin gene allows cells to grow vigorously in a new environment.IMPORTANCEThermococcus kodakarensispossesses two group II chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively. CpkA might act as an “adaptive allele” to adapt to cooler environments. In this study, we compared the last 20 amino acids within the C termini of the chaperonins and found a clear correlation between the CpkA-type chaperonin gene copy number and growth temperature. Furthermore, we introduced single mutations into the CpkA C-terminal region to clarify its role in cold adaptation, and we showed that a single base substitution allowed the organism to adapt to a lower temperature. The present data suggest that hyperthermophiles have evolved by obtaining mutations in chaperonins that allow them to adapt to a colder environment.

scholarly journals Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

2014 ◽  
Vol 82 (11) ◽  
pp. 4758-4766 ◽  
Author(s):  
Megan M. Jones ◽  
Antoinette Johnson ◽  
Mary Koszelak-Rosenblum ◽  
Charmaine Kirkham ◽  
Aimee L. Brauer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Respiratory Tract ◽  
Moraxella Catarrhalis ◽  
Minimal Medium ◽  
Substrate Binding ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Content Type ◽  
Oligopeptide Permease ◽  
Substrate Binding Protein

ABSTRACTMoraxella catarrhalisis a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality.M. catarrhalishas a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival.M. catarrhalishas a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB,oppC,oppD,oppF, andoppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which theoppBCDFAgene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth ofM. catarrhalisis important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-typeM. catarrhaliswas observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requiresl-arginine. AnoppAknockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.

scholarly journals Plasmid Localization and Organization of Melamine Degradation Genes in Rhodococcus sp. Strain Mel

2011 ◽  
Vol 78 (5) ◽  
pp. 1397-1403 ◽  
Author(s):  
Anthony G. Dodge ◽  
Lawrence P. Wackett ◽  
Michael J. Sadowsky
Keyword(s):  
454 Pyrosequencing ◽  
Minimal Medium ◽  
Doubling Time ◽  
Cyanuric Acid ◽  
Acid Hydrolase ◽  
Content Type ◽  
N Source ◽  
Genes Encoding ◽  
Rhodococcus Sp ◽  
Roche 454

ABSTRACTRhodococcussp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase genetrzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ∼265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. TheRhodococcussp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired.

scholarly journals Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Keiji Kiyoshi ◽  
Sohei Kawashima ◽  
Kosuke Nobuki ◽  
Toshimori Kadokura ◽  
Atsumi Nakazato ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Viability ◽  
Spore Formation ◽  
Butanol Production ◽  
Metabolomic Analysis ◽  
Primary Metabolites ◽  
Content Type ◽  
Atp Levels ◽  
Wide Range ◽  
Multiple Primary

ABSTRACT We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels. IMPORTANCE Metabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.

scholarly journals Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

2015 ◽  
Vol 83 (3) ◽  
pp. 1190-1198 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Uma M. Sharma ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Secretion System ◽  
Terminal Region ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv

Coxiella burnetiicauses human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages,C. burnetiiuses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100C. burnetiiDot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in allC. burnetiiisolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizingproteinA). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function duringC. burnetiiinfection.

scholarly journals A novel inhibitor of indole-3-glycerol phosphate synthase with activity against multidrug-resistant Mycobacterium tuberculosis

FEBS Journal ◽  
2008 ◽  
Vol 276 (1) ◽  
pp. 144-154 ◽  
Author(s):  
Hongbo Shen ◽  
Feifei Wang ◽  
Ying Zhang ◽  
Qiang Huang ◽  
Shengfeng Xu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistant ◽  
Glycerol Phosphate ◽  
Phosphate Synthase

scholarly journals The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content

2021 ◽  
Vol 87 (6) ◽  
Author(s):  
Katsuya Fuchino ◽  
Helena Chan ◽  
Ling Chin Hwang ◽  
Per Bruheim
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
Cell Size ◽  
Dna Content ◽  
Bacterial Cell ◽  
Zymomonas Mobilis ◽  
Biofuel Production ◽  
Public Attention ◽  
Content Type ◽  
Cell Organization

ABSTRACT The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilis. IMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker’s yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

scholarly journals Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

2016 ◽  
Vol 82 (10) ◽  
pp. 3022-3031 ◽  
Author(s):  
Ayako Fujiwara ◽  
Katsuhiro Kawato ◽  
Saori Kato ◽  
Kiyoshi Yasukawa ◽  
Ryota Hidese ◽  
...  
Keyword(s):  
Noise Reduction ◽  
Dna Polymerase ◽  
Biological Activities ◽  
Genetic Diagnosis ◽  
Gc Content ◽  
Dna Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Thermococcus Kodakarensis

ABSTRACTDNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, theEuryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeonThermococcus kodakarensis(Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined.Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5′ overhung, 3′ overhung, and blunt end) at 50°C.Tk-EshA unwound forked and 3′ overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers whenTk-EshA was added to a PCR mixture. To examine the effect ofTk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence ofTk-EshA. In contrast, noise DNAs were eliminated in the presence ofTk-EshA. Noise reduction byTk-EshA was also confirmed whenTaqDNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated duringtoxAgene amplification fromPseudomonas aeruginosaDNA, which possesses a high GC content (69%).Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs duringtoxAamplification.Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR.IMPORTANCEPCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostableEuryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition ofTk-EshA has reduced noise DNAs in PCR.

scholarly journals Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Volker Winstel ◽  
Patricia Sanchez-Carballo ◽  
Otto Holst ◽  
Guoqing Xia ◽  
Andreas Peschel
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biosynthesis Pathway ◽  
Glycerol Phosphate ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Wall Teichoic Acids

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

scholarly journals The Essential Genome of Burkholderia cenocepacia H111

2017 ◽  
Vol 199 (22) ◽  
Author(s):  
Steven Higgins ◽  
Maria Sanchez-Contreras ◽  
Stefano Gualdi ◽  
Marta Pinto-Carbó ◽  
Aurélien Carlier ◽  
...  
Keyword(s):  
Minimal Medium ◽  
Essential Genes ◽  
Burkholderia Cenocepacia ◽  
Chromosome 1 ◽  
Biological Research ◽  
Chromosome 2 ◽  
Powerful Method ◽  
Rich Medium ◽  
Content Type ◽  
Autonomous Growth

ABSTRACT The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera. IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of “core essential genes” that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

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