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Asset Tag: de novo assembly

September 22, 2019

Genomic analysis of a pan-resistant isolate of Klebsiella pneumoniae, United States 2016.

Antimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusualKlebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S.The isolate harbored four known beta-lactamase genes, including plasmid-mediatedblaNDM-1andblaCMY-6, as well as chromosomalblaCTX-M-15andblaSHV-28, which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the firstK. pneumoniaeisolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline. IMPORTANCE Antimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. AKlebsiella pneumoniaestrain that was nonsusceptible to all tested antibiotics was isolated from a U.S.Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.

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September 22, 2019

Chinook salmon (Oncorhynchus tshawytscha) genome and transcriptome.

When unifying genomic resources among studies and comparing data between species, there is often no better resource than a genome sequence. Having a reference genome for the Chinook salmon (Oncorhynchus tshawytscha) will enable the extensive genomic resources available for Pacific salmon, Atlantic salmon, and rainbow trout to be leveraged when asking questions related to the Chinook salmon. The Chinook salmon’s wide distribution, long cultural impact, evolutionary history, substantial hatchery production, and recent wild-population decline make it an important research species. In this study, we sequenced and assembled the genome of a Chilliwack River Hatchery female Chinook salmon (gynogenetic and homozygous at all loci). With a reference genome sequence, new questions can be asked about the nature of this species, and its role in a rapidly changing world.

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September 22, 2019

Characterization of two novel bacteriophages infecting multidrug-resistant (MDR) Acinetobacter baumannii and evaluation of their therapeutic efficacy in vivo.

Acinetobacter baumannii is emerging as a challenging nosocomial pathogen due to its rapid evolution of antibiotic resistance. We report characterization of two novel bacteriophages, PBAB08 and PBAB25, infecting clinically isolated, multidrug-resistant (MDR) A. baumannii strains. Both phages belonged to Myoviridae of Caudovirales as their morphology observed under an electron microscope. Their genomes were double stranded linear DNAs of 42,312 base pairs and 40,260 base pairs, respectively. The two phages were distinct from known Acinetobacter phages when whole genome sequences were compared. PBAB08 showed a 99% similarity with 57% sequence coverage to phage AB1 and PBAB25 showed a 97% similarity with 78% sequence coverage to phage IME_AB3. BLASTN significant alignment coverage of all other known phages were <30%. Seventy six and seventy genes encoding putative phage proteins were found in the genomes of PBAB08 and PBAB25, respectively. Their genomic organizations and sequence similarities were consistent with the modular theory of phage evolution. Therapeutic efficacy of a phage cocktail containing the two and other phages were evaluated in a mice model with nasal infection of MDR A. baumannii. Mice treated with the phage cocktail showed a 2.3-fold higher survival rate than those untreated in 7 days post infection. In addition, 1/100 reduction of the number of A. baumannii in the lung of the mice treated with the phage cocktail was observed. Also, inflammatory responses of mice which were injected with the phage cocktail by intraperitoneal, intranasal, or oral route was investigated. Increase in serum cytokine was minimal regardless of the injection route. A 20% increase in IgE production was seen in intraperitoneal injection route, but not in other routes. Thus, the cocktail containing the two newly isolated phages could serve as a potential candidate for therapeutic interventions to treat A. baummannii infections.

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September 22, 2019

Inferring the minimal genome of Mesoplasma florum by comparative genomics and transposon mutagenesis.

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

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September 22, 2019

Genomic architecture of haddock (Melanogrammus aeglefinus) shows expansions of innate immune genes and short tandem repeats.

Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group.Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) – a relative of Atlantic cod – using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes.The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod – but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates.

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September 22, 2019

The genome of Ectocarpus subulatus highlights unique mechanisms for stress tolerance in brown algae

Brown algae are multicellular photosynthetic organisms belonging to the stramenopile lineage. They are successful colonizers of marine rocky shores world-wide. The genus Ectocarpus, and especially strain Ec32, has been established as a genetic and genomic model for brown algae. A related species, Ectocarpus subulatus Kuetzing, is characterized by its high tolerance of abiotic stress. Here we present the genome and metabolic network of a haploid male strain of E. subulatus, establishing it as a comparative model to study the genomic bases of stress tolerance in Ectocarpus. Our analyses indicate that E. subulatus has separated from Ectocarpus sp. Ec32 via allopatric speciation. Since this event, its genome has been shaped by the activity of viruses and large retrotransposons, which in the case of chlorophyll-binding proteins, may be related to the expansion of this gene family. We have identified a number of further genes that we suspect to contribute to stress tolerance in E. subulatus, including an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defense compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. However, 96% of genes that differed between the two examined Ectocarpus species, as well as 90% of genes under positive selection, were found to be lineage-specific and encode proteins of unknown function. This underlines the uniqueness of brown algae with respect to their stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies.

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September 22, 2019

Genomic structural variations affecting virulence during clonal expansion of Pseudomonas syringae pv. actinidiae biovar 3 in Europe.

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

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September 22, 2019

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm.

The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO[Formula: see text]) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59.The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO[Formula: see text], nitrite (NO[Formula: see text]) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures.During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO[Formula: see text] under oxic culture conditions, and has a 24-h lag time before growth and NO[Formula: see text] reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO[Formula: see text] as it carries a gene encoding a NirK-type NO[Formula: see text] reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process.These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

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September 22, 2019

The Phytophthora cactorum genome provides insights into the adaptation to host defense compounds and fungicides.

Phytophthora cactorum is a homothallic oomycete pathogen, which has a wide host range and high capability to adapt to host defense compounds and fungicides. Here we report the 121.5?Mb genome assembly of the P. cactorum using the third-generation single-molecule real-time (SMRT) sequencing technology. It is the second largest genome sequenced so far in the Phytophthora genera, which contains 27,981 protein-coding genes. Comparison with other Phytophthora genomes showed that P. cactorum had a closer relationship with P. parasitica, P. infestans and P. capsici. P. cactorum has similar gene families in the secondary metabolism and pathogenicity-related effector proteins compared with other oomycete species, but specific gene families associated with detoxification enzymes and carbohydrate-active enzymes (CAZymes) underwent expansion in P. cactorum. P. cactorum had a higher utilization and detoxification ability against ginsenosides-a group of defense compounds from Panax notoginseng-compared with the narrow host pathogen P. sojae. The elevated expression levels of detoxification enzymes and hydrolase activity-associated genes after exposure to ginsenosides further supported that the high detoxification and utilization ability of P. cactorum play a crucial role in the rapid adaptability of the pathogen to host plant defense compounds and fungicides.

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September 22, 2019

Complete genomic analysis of a Salmonella enterica Serovar Typhimurium isolate cultured from ready-to-eat pork in China carrying one large plasmid containing mcr-1.

One mcr-1-carrying ST34-type Salmonella Typhimurium WW012 was cultured from 3,200 ready-to-eat (RTE) pork samples in 2014 in China. Broth dilution method was applied to obtain the antimicrobial susceptibility of Salmonella Typhimurium WW012. Broth matting assays were carried out to detect transferability of this phenotype and whole-genome sequencing was performed to analyze its genomic characteristic. Thirty out of 3,200 RTE samples were positive for Salmonella and the three most frequent serotypes were identified as S. Derby (n = 8), S. Typhimurium (n = 6), and S. Enteritidis (n = 6). One S. Typhimurium isolate (S. Typhimurium WW012) cultured from RTE prepared pork was found to contain the mcr-1 gene. S. Typhimurium WW012 expressed a level of high resistance to seven different antimicrobial compounds in addition to colistin (MIC = 8 mg/L). A single plasmid, pWW012 (151,609-bp) was identified and found to be of an IncHI2/HI2A type that encoded a mcr-1 gene along with six additional antimicrobial resistance genes. Plasmid pWW012 contained an IS30-mcr-1-orf-orf-IS30 composite transposon that can be successfully transferred to Escherichia coli J53. When assessed further, the latter demonstrated considerable similarity to three plasmids pHYEC7-mcr-1, pSCC4, and pHNSHP45-2, respectively. Furthermore, plasmid pWW012 also contained a multidrug resistance (MDR) genetic structure IS26-aadA2-cmlA2-aadA1-IS406-sul3-IS26-dfrA12-aadA2-IS26, which showed high similarity to two plasmids, pHNLDF400 and pHNSHP45-2, respectively. Moreover, genes mapping to the chromosome (4,991,167-bp) were found to carry 28 mutations, related to two component regulatory systems (pmrAB, phoPQ) leading to modifications of lipid A component of the lipopolysaccharide structure. Additionally, one mutation (D87N) in the quinolone resistance determining region (QRDR) gene of gyrA was identified in this mcr-1 harboring S. Typhimurium. In addition, various virulence factors and heavy metal resistance-encoding genes were also identified on the genome of S. Typhimurium WW012. This is the first report of the complete nucleotide sequence of mcr-1-carrying MDR S. Typhimurium strain from RTE pork in China.

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September 22, 2019

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (Camellia sinensis)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.

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September 22, 2019

The complete chloroplast genome of Chrysanthemum boreale (Asteraceae)

Chrysanthemum boreale is a perennial plant in the Asteraceae family that is native to eastern Asia and has both ornamental and herbal uses. Here, we determined the complete chloroplast genome sequence for C. boreale using long-read sequencing. The chloroplast genome was 151,012?bp and consisted of a large single copy (LSC) region (82,817?bp), a small single copy (SSC) region (18,281?bp) and two inverted repeats (IRs) (24,957?bp). It was predicted to contain 131 genes, including 87 protein-coding genes, eight rRNAs and 46 tRNAs. Phylogenetic analysis of chloroplast genomes clustered C. boreale with other Chrysanthemum and Asteraceae species.

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September 22, 2019

Evaluation of WGS based approaches for investigating a food-borne outbreak caused by Salmonella enterica serovar Derby in Germany.

In Germany salmonellosis still represents the 2nd most common bacterial foodborne disease. The majority of infections are caused by Salmonella (S.) Typhimurium and S. Enteritidis followed by a variety of other broad host-range serovars. Salmonella Derby is one of the five top-ranked serovars isolated from humans and it represents one of the most prevalent serovars in pigs, thus bearing the potential risk for transmission to humans upon consumption of pig meat and products thereof. From November 2013 to January 2014 S. Derby caused a large outbreak that affected 145 primarily elderly people. Epidemiological investigations identified raw pork sausage as the probable source of infection, which was confirmed by microbiological evidence. During the outbreak isolates from patients, food specimen and asymptomatic carriers were investigated by conventional typing methods. However, the quantity and quality of available microbiological and epidemiological data made this outbreak highly suitable for retrospective investigation by Whole Genome Sequencing (WGS) and subsequent evaluation of different bioinformatics approaches for cluster definition. Overall the WGS-based methods confirmed the results of the conventional typing but were of significant higher discriminatory power. That was particularly beneficial for strains with incomplete epidemiological data. For our data set both, single nucleotide polymorphism (SNP)- and core genome multilocus sequence typing (cgMLST)-based methods proved to be appropriate tools for cluster definition. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019

The antibody loci of the domestic goat (Capra hircus).

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

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September 22, 2019

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

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