Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Transcriptomic Analysis of Listeria monocytogenes in Response to Bile Under Aerobic and Anaerobic Conditions

Listeria monocytogenes is a gram-positive facultative anaerobic bacterium that causes the foodborne illness listeriosis. The pathogenesis of this bacterium depends on its survival in anaerobic, acidic, and bile conditions encountered throughout the gastrointestinal (GI) tract. This transcriptomics study was conducted to analyze the differences in transcript levels produced under conditions mimicking the GI tract. Changes in transcript levels were analyzed using RNA isolated from L. monocytogenes strain F2365 at both aerobic and anaerobic conditions, upon exposure to 0 and 1% bile at acidic and neutral pH. Transcripts corresponding to genes responsible for pathogenesis, cell wall associated proteins, DNA repair, transcription factors, and stress responses had variations in levels under the conditions tested. Upon exposure to anaerobiosis in acidic conditions, there were variations in the transcript levels for the virulence factors internalins, listeriolysin O, etc., as well as many histidine sensory kinases. These data indicate that the response to anaerobiosis differentially influences the transcription of several genes related to the survival of L. monocytogenes under acidic and bile conditions. Though further research is needed to decipher the role of oxygen in pathogenesis of L. monocytogenes, these data provide comprehensive information on how this pathogen responds to the GI tract.

Effect of terahertz radiation on intermolecular interactions of albumin under aerobic and anaerobic conditions

The effect of THz radiation on coupling between albumin molecules under aerobic and anaerobic conditions is assessed through an EPR-quantified procedure. Rearrangements induced by terahertz radiation are found to affect the hydrogen bonding network causing an increase in the rate of bimolecular interactions. Molecular mechanism of the observed effect is considered involving the interactions between the functional groups of albumin molecules. Irradiation causes conformational changes in albumin molecules, which involves changes in the steric states of molecules. These rearrangements hinder or simplify the adsorption of specific agents on the reactive sites of albumin molecules.

An Environment Transitional Zone Buffers Peatlands Carbon Loss

An environment transitional zone (ETzone) is usually deemed as a hotspot in biogeochemical cycle, but little is known about its response to climate change. A typical ETzone develops at the subsurface of peatland after experiencing long-term water table fluctuation, characterized by alternative aerobic and anaerobic conditions. By an extensive incubation, we found that the CO2 emission at this ETzone was 28.31 ± 3.55 μg g-1 d-1, 41.6% and 34.4% lower than the upper (aerobic) and lower (anaerobic) layers, respectively. Moreover, with a lowest Q10 of 1.37, its CO2 emission was also the least warming-responsive, which could reduce 33.7% CO2 loss in warming scenario. This result clearly revealed that the ETzone worked as a buffer to retard carbon loss, rather than a hotspot. Surprisingly, this buffer capacity of ETzone was easily collapse if being primed by fresh carbon. Therefore, maintaining a relatively stable ETzone is critical for protecting peatland carbon stock, and the priority is to block priming effect through maintaining an intact vegetation composition.

Anaerobic osteomyelitis

The prevalence and role of anaerobic bacteria in bone infection were investigated in this prospective study on 134 cases with pyogenic osteomyelitis. Specimens were inoculated immediately in the operating theatre or in the ward and incubated under aerobic and anaerobic conditions without using transport media. Anaerobic organisms were isolated from 39 of the 134 cases [29%] of all types of pyogenic osteomyelitis. The total number of aerobic and anaerobic isolates was 224, of which 50 were anaerobes [22%]. Syringe-aspirated specimens were better than swab specimens for the isolation of anaerobes. Anaerobes were mostly isolated from osteomyelitis cases of long duration

Cultivable Oral Microbiota in Puppies

The oral microbiota has been shown to be different in children born by caesarean section and delivered vaginally. The aim of this study was to investigate the oral microbial diversity in healthy puppies and to determine whether the birth mode affects the composition of the oral microbiota. A total of 19 puppies from 4 dams were included in the study. The puppies were divided into two groups depending on the birth mode, vaginal delivery (vaginal born VB) or caesarean delivery (caesar-ean section CS). On the seventh day after birth, swabs of the oral cavity were taken. All samples were analysed by bacteriological cultivation under aerobic and anaerobic conditions. Bacterial colonies were identified by Sanger sequencing of 16S rRNA. A total of 64 bacterial strains belonging to 10 genera were obtained from the oral swabs. The genera Staphylococcus (30.23 % VB and 47.62 % CS) and Enterococcus (25.58 % VB and 33.33 % CS) were the most abundant in both groups. The genera Escherichia (18.60 %) and Enterobacter (16.28 %) were largely present in puppies delivered vaginally, they were not found in puppies born by caesarean section. The other detected genera were present at lower proportions (< 5 %) and varied between the groups. The oral micro-biota of the puppies in the litter was similar, but differed between litters and between groups. Based on these results, we can assume that the birth mode affects the oral microbiota of puppies.

Effect of Addition of Tryptophan on Aggregation of Apo-α-Lactalbumin Induced by UV-Light

UV-B illumination facilitates aggregation of alpha-lactalbumin (α-LA) by intramolecular disulfide bond cleavage followed by intermolecular thiol-disulfide exchange reactions. However, long term exposure to UV-B illumination may induce undesired oxidative modifications of amino acid residues in the protein. The purpose of this study was to examine the effect of UV-induced aggregation of apo-α-LA (a calcium-depleted form of α-LA) under aerobic and anaerobic conditions and by addition of tryptophan (Trp) as a photosensitizer. The addition of Trp to apo-α-LA illuminated under anaerobic conditions facilitated the highest level of free thiol release and disulfide-mediated aggregation as compared to without addition of Trp under both anaerobic and aerobic conditions. Addition of Trp under aerobic condition resulted in the lowest level of free thiols and disulfide-mediated aggregation and the aerobic conditions caused oxidation of the free Trp with formation of kynurenine and 5-hydroxy-Trp. Minor levels of the Trp oxidation product, 3-hydroxy-kynurenine (2% converted from Trp), was formed in apo-α-LA with added Trp under both aerobic and anaerobic conditions after UV-B treatment.