Ingestion of Nylon 11 Polymers by the Mealworm (Tenebrio molitor) Beetle and Subsequent Enrichment of Monomer-Metabolizing Bacteria in Fecal Microbiome.

BACKGROUND: Nylon 11 is a synthetic plastic widely used in commercial products such as tubing for automobiles, offshore oilfields, and medical devices. An increasing amount of nylon and other plastic wastes have been released into various environments, posing ecological threats. The biodegradation of bundled nylon polymers has been considered impossible due to their crystalline structures. METHODS: Nylon 11 film was created and incubated with adult mealworms. The mass, as well as structures, of nylon 11 films at pre- and post-incubation with beetles were compared. The number of nylon 11 monomer degrading bacteria in feces were determined by culture-dependent approach. The t-test was utilized to examine the statistical significance. RESULTS: We discovered that adult mealworm (Tenebrio molitor) beetle can ingest nylon 11 when stretched thin. The microscopic observation of their feces did not identify the presence of large fragments of nylon 11. The analysis of fecal bacteria revealed that while the total number of culturable bacteria did not change significantly, the number of 11-aminoundecanoic acid-metabolizing bacteria increased by 10,000-fold. CONCLUSIONS: Our results suggest that bundled nylon 11 polymers were fragmented into smaller pieces, including monomeric units (11-aminoundecanoic acid) by adult mealworm. The monomers seem to have supported the proliferation of gut microbial communities capable of utilizing 11-aminoundecanoic acid as a carbon and nitrogen source. Our work implies the potential use of the mealworm beetle as a means to fragment nylon polymers for remediation applications.

Formation of unique T-shape budding and differential impacts of low surface water on Bacillus mycoides rhizoidal colony.

Bacillus mycoides Ko01 strain grows rapidly and forms extensive rhizoidal colonies on hard agar despite limited surface water availability. The agar concentrations affect the handedness of the colonies as well as other colony architectures. In this study, we found that the local curvature of cell chains in the developing colonies did not vary based on the agar concentration, while concentration does affect the handedness of chirality at the macroscale. This result suggests independence between the microscale filament curvature and macroscale colony chirality. In addition, we discovered a novel microscopic property of cells that has not been observed before: T-shaped budding under extremely low surface water availability conditions. We propose that this feature gives rise to chaotic colony morphology. Together with bundling of chains, cells form a unique set of spatial arrangements under different surface water availability. These properties appear to impact the structural features of thick tendrils, and thereby the overall morphology of colonies. Our study provides additional insights as to how bacteria proliferate, spread, and develop macroscale colony architecture under water-limited conditions.

Discovery of Nylon 11 ingestion by mealworm (Tenebrio molitor) larvae and detection of monomer-degrading bacteria in gut microbiota.

Nylon 11, which can be found in many commercial products, is a synthetic plastic that has previously been considered non-biodegradable. Increasing nylon 11 and other plastics in landfills and in the environment pose an environmental concern. Recent studies on plastic biodegradation revealed that initial mechanical fragmentations increase the rate of degradation. In this study, we discovered that the larvae of mealworm (Tenebrio molitor) can masticate nylon 11 film at the rate of 0.25 ± 0.07 mg per fifty larvae per day. The body mass of larvae did not differ from that of starvation control while feeding on nylon 11. Comparison of gut microbiota in nylon-fed and starving larvae showed a shift in composition. There was a significant variation in community composition among the nylon 11-fed experimental groups, suggesting that many organisms are capable of metabolizing nylon 11 fragments and/or possess a growth advantage in a nylon-fed gut environment. We also discovered that a significant fraction of gut microbiome of control larvae is capable of metabolizing nylon 11 monomer (11-aminoundecanoic acid) even in the absence of prior exposure to nylon 11. This is the first study demonstrating ingestion of nylon polymers by invertebrates, and our results suggest the potential of mealworm larvae for nylon 11 biodegradation applications.

Cellular Memory of HipA-Induced Growth Arrest: The Length of Cell Growth Arrest Becomes Shorter for Each Successive Induction.

Toxin-antitoxin (TA) systems are genetic modules found commonly in bacterial genomes. HipA is a toxin protein encoded from the hipBA TA system in the genome of Escherichia coli. Ectopic expression of hipA induces cell growth arrest. Unlike the cell growth arrest caused by other TA toxins, cells resume growth from the HipA-induced cell growth arrest phase after a defined period of time. In this article, we describe the change in the length of growth arrest while cells undergo repeated cycles of hipA induction, growth arrest and regrowth phases. In the multiple conditions tested, we observed that the length of growth arrest became successively shorter for each round of induction. We verified that this was not due to the appearance of HipA-resistant mutants. Additionally, we identified conditions, such as the growth phase of the starting culture and growth vessels, that alter the length of growth arrest. Our results showed that the length of HipA-induced growth arrest was dependent on environmental factors-in particular, the past growth environment of cells, such as a previous hipA induction. These effects lasted even after multiple rounds of cell divisions, indicating the presence of cellular "memory" that impacts cells' response to HipA-induced toxicity.

Isolation and genomic analysis of 11-aminoundecanoic acid-degrading bacterium Pseudomonas sp. JG-B from nylon 11 enrichment culture.

Nylon 11 is a polymer synthesized from 11-aminoundecanoic acid, and widely used in commercial manufacturing. In this study, we describe the isolation of the first organism capable of metabolizing 11-aminoundecanoic acid from nylon 11 enrichment culture. The strain shows rapid growth on 11-aminoundecanoic acid as a sole source of carbon, nitrogen, and energy. Furthermore, the genome sequence of strain JG-B was deciphered and shown to belong to genus Pseudomonas. Many genes encoding putative extracellular hydrolases, as well as homologues of nylon 6 hydrolases (NylB and NylA) were identified, suggesting the metabolic versatility and possibility that this organism could also depolymerase nylon 11 polymers.

Comparative Proteomic Analysis of Propane Metabolism in Mycobacterium sp. Strain ENV421 and Rhodococcus sp. Strain ENV425.

While growing on propane as a sole source of carbon, many strains cometabolically degrade environmental pollutants, such as ethers and chlorinated hydrocarbons. To gain insights into the molecular basis behind such a high metabolic versatility of propanotrophs, we examined the propane-inducible protein expression patterns of 2 soil actinobacteria that are known to degrade a variety of ethers (i.e., Mycobacterium sp. strain ENV421 and Rhodococcus sp. strain ENV425). In both strains, soluble diiron monooxygenase(s), that would catalyze the first step of the pathway, were induced by propane. However, despite their phylogenetic similarity, different sets of additional putative propane oxygenases (e.g., cytochrome P450 and particulate methane monooxygenases) were overexpressed in the 2 strains. They also diverged in the expression of enzymes responsible for downstream reactions. This study revealed a diversity of expression of putative propane oxygenases, which may be responsible for xenobiotic degradation, as well as a variety of metabolic pathways for propane in these bacterial species.

Genomic Analysis of Propane Metabolism in Methyl Tert-Butyl Ether-Degrading Mycobacterium Sp. Strain ENV421.

Methyl tert-butyl ether (MTBE) is a ground water contaminant with plausible carcinogenic properties. Mycobacterium sp. strain ENV421 cometabolically degrades MTBE and other ethers during the growth on propane as a carbon source. In this study, the 6.2 Mb genome of strain ENV421 was deciphered. The genome sequence revealed the presence of numerous putative propane catabolic genes including genes encoding hydrocarbon oxygenases and short chain alcohol dehydrogenases. These data provide the basis for the elucidation of propane metabolic pathways in strain ENV421 and its application for the remediation of ground water contaminated with toxic ethers.

Draft Genome Sequence of a Propanotroph, Rhodococcus sp. Strain ENV425, Capable of Degrading Methyl tert-Butyl Ether and N-Nitrosodimethylamine.

In this study, the draft genome of Rhodococcus sp. strain ENV425 was determined. The propane-grown strain ENV425 cometabolically degrades environmental contaminants such as methyl tert-butyl ether and N-nitrosodimethylamine. The sequence revealed the presence of multiple hydrocarbon metabolic genes that could play pivotal roles in the biodegradation of pollutants.

Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity.

Protein translation is the most common target of toxin-antitoxin system (TA) toxins. Sequence-specific endoribonucleases digest RNA in a sequence-specific manner, thereby blocking translation. While past studies mainly focused on the digestion of mRNA, recent analysis revealed that toxins can also digest tRNA, rRNA and tmRNA. Purified toxins can digest single-stranded portions of RNA containing recognition sequences in the absence of ribosome in vitro. However, increasing evidence suggests that in vivo digestion may occur in association with ribosomes. Despite the prevalence of recognition sequences in many mRNA, preferential digestion seems to occur at specific positions within mRNA and also in certain reading frames. In this review, a variety of tools utilized to study the nuclease activities of toxins over the past 15 years will be reviewed. A recent adaptation of an RNA-seq-based technique to analyze entire sets of cellular RNA will be introduced with an emphasis on its strength in identifying novel targets and redefining recognition sequences. The differences in biochemical properties and postulated physiological roles will also be discussed.

Switching of the rotational direction of rhizoidal colonies in a newly isolated Bacillus mycoides strain Ko01.

Bacillus mycoides are known to form rhizoidal colonies on solid medium. In this study, a new strain of B. mycoides, strain Ko01, was isolated from soil. Genetic and growth patterns indicated that this strain belongs to subgroup II of the B. cereus group. Strain Ko01 forms extensive rhizoidal colonies with predictable directions of rotation. The concentration of the agar, and not the chemical composition, altered the direction of the colony rotation, switching from counterclockwise to clockwise. Agar concentration-dependent switching of rotation direction was unique to strain Ko01 and was not seen in colonies of other B. mycoides strains that were tested. Factors affecting colony chirality patterns appeared to be variable among B. mycoides strains. This feature can be used for the classification of B. mycoides strains.

ydfD encodes a novel lytic protein in Escherichia coli.

Bacteria carry a number of genes that cause cell growth arrest or cell lysis upon expression. Notably, defective prophages retain many lysis proteins. Here, we identified a novel lytic gene, ydfD, on the Qin prophage segment of the Escherichia coli genome. YdfD lyses 99.9% of cells within 2 h of its induction. The co-expression of the upstream gene, dicB, encoding a cell division inhibitor, as well as sulA, encoding another cell division inhibitor, abolished YdfD-induced cell lysis. These results imply that YdfD-induced lysis is a cell division-dependent event. We further found that by deleting the hydrophobic 22-residue N-terminal domain, the resulting 42-residue C-terminal domain was still toxic to cause cell lysis. We propose that YdfD, associated with the cytoplasmic membrane, inhibits an essential cellular process(s).

Draft Genome Sequence of the Versatile Alkane-Degrading Bacterium Aquabacterium sp. Strain NJ1.

The draft genome sequence of a soil bacterium, Aquabacterium sp. strain NJ1, capable of utilizing both liquid and solid alkanes, was deciphered. This is the first report of an Aquabacterium genome sequence.

Transcriptional repressor HipB regulates the multiple promoters in Escherichia coli.

HipB is a DNA-binding protein in Escherichia coli and negatively regulates its own promoter by binding to the palindromic sequences [TATCCN8GGATA (N represents any nucleotides)] on the hipBA promoter. For such sequences, bioinformatic analysis revealed that there are a total of 39 palindromic sequences (TATCCN(x)GGATA: N is any nucleotides and x is the number of nucleotides from 1 to 30) in the promoter regions of 33 genes on the E. coli genome. Notably, eutH and fadH have two and three TATCCN(x)GGATA palindromic sequences located in their promoters, respectively. Another significant finding was that a palindromic sequence was also identified in the promoter region of hipAB locus, known to be involved in the RelA-dependent persister cell formation in bacteria. Here, we demonstrated that HipB binds to the palindromic structures in the eutH, fadH, as well as the relA promoter regions and represses their expressions. We further demonstrated that HipA enhances the repression of the relA promoter activity by HipB. This effect was not observed with D291A HipA mutant which was previously shown to lack an ability to interact with HipB, indicating that HipA enhances the HipB's repressor activity through direct interaction with HipB.

Biodegradation of tetrahydrofuran and 1,4-dioxane by soluble diiron monooxygenase in Pseudonocardia sp. strain ENV478.

1,4-Dioxane is an important groundwater contaminant. Pseudonocardia sp. strain ENV478 degrades 1,4-dioxane via cometabolism after the growth on tetrahydrofuran (THF) and other carbon sources. Here, we have identified a THF monooxygenase (thm) in ENV478. The thm genes are transcribed constitutively and are induced to higher levels by THF. Decreased translation of the thmB gene encoding one of the monooxygenase subunits by antisense RNA resulted in the loss of its ability to degrade THF and 1,4-dioxane. This is the first study to link thm genes to THF degradation, as well as the cometabolic oxidation of 1,4-dioxane.

YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli.

All free-living bacteria carry the toxin-antitoxin (TA) systems controlling cell growth and death under stress conditions. YeeU-YeeV (CbtA) is one of the Escherichia coli TA systems, and the toxin, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ. Here, we demonstrate that the antitoxin, YeeU, is a novel type of antitoxin (type IV TA system), which does not form a complex with CbtA but functions as an antagonist for CbtA toxicity. Specifically, YeeU was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro. Surprisingly, YeeU neutralized not only the toxicity of CbtA but also the toxicity caused by other inhibitors of MreB and FtsZ, such as A22, SulA and MinC, indicating that YeeU-induced bundling of MreB and FtsZ has an intrinsic global stabilizing effect on their homeostasis. Here we propose to rename YeeU as CbeA for cytoskeleton bundling-enhancing factor A.

A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli.

Nearly all free-living bacteria carry toxin-antitoxin (TA) systems on their genomes, through which cell growth and death are regulated. Toxins target a variety of essential cellular functions, including DNA replication, translation, and cell division. Here, we identified a novel toxin, YgfX, on the Escherichia coli genome. The toxin, consisting of 135 residues, is composed of the N-terminal membrane domain, which encompasses two transmembrane segments, and the C-terminal cytoplasmic domain. Upon YgfX expression, the cells were initially elongated and then the middle portion of the cells became inflated to form a lemon shape. YgfX was found to interact with MreB and FtsZ, two essential cytoskeletal proteins in E. coli. The cytoplasmic domain [YgfX(C)] was found to be responsible for the YgfX toxicity, as purified YgfX(C) was found to block the polymerization of FtsZ and MreB in vitro. YgfY, located immediately upstream of YgfX, was shown to be the cognate antitoxin; notably, YgfX is the first membrane-associating toxin in bacterial TA systems. We propose to rename the toxin and the antitoxin as CptA and CptB (for Cytoskeleton Polymerization inhibiting Toxin), respectively.

Aerobic biodegradation of N-nitrosodimethylamine by the propanotroph Rhodococcus ruber ENV425.

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [(14)C]NDMA to (14)CO(2), growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.

Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478.

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for > 80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.