Comprehensive Genomics and Proteomics Analysis Reveals the Multiple Response Strategies of Endophytic Bacillus sp. WR13 to Iron Limitation.

Iron (Fe) is an important metal element for the growth of bacteria. Many bacteria respond to Fe limitation through a variety of strategies. We previously isolated an endophyte Bacillus sp. WR13 from wheat root. However, whether and how this strain can cope with Fe-deficient environments remains unclear. In this study, the growth of WR13 under Fe starvation was investigated, and the underlying mechanisms of WR13 in response to Fe starvation were elucidated via genomics and iTRAQ-based proteomics. Under Fe limitation, WR13 showed a growth pattern similar to that of Fe sufficiency. Genomics analysis demonstrated that WR13 had gene clusters related to siderophore synthesis (dhbACEBF), transportation (bcbE), uptake (feuABC-yusV) and hydrolysis (besA). These genes were significantly up-regulated in Fe-starved WR13, which resulted in more siderophore production. Proteomics data revealed that many Fe-containing proteins such as ACO, HemQ, ferredoxin, CNP, and SufD were significantly reduced under Fe limitation. Meanwhile, significant decreases in many proteins involved in glycolysis, TCA cycle, pentose phosphate pathway; asparagine, glutamine, methionine, and serine metabolism; and phospholipid hydrolysis were also observed. Overall, this study shows that Bacillus sp. WR13 was able to respond to Fe limitation via multiple strategies and provides a theoretical basis for the application of WR13 in Fe-deficient soil.

Harnessing Native Bacillus spp. for Sustainable Wheat Production.

The genus Bacillus has been widely applied in contemporary agriculture as an environmentally-friendly biological agent. However, the real effect of commercial Bacillus-based fertilizers and pesticides varies immensely in the field. To harness Bacillus for efficient wheat production, we reviewed the diversity, functionality, and applicability of wheat-associated native Bacillus for the first time. Our main findings are: (i) Bacillus spp. inhabit the rhizosphere, root, stem, leaf, and kernel of wheat; (ii) B. subtilis and B. velezensis are the most widely endophytic species that can be isolated from both below and aboveground tissues; (iii) major functions of these representative strains are promotion of plant growth and alleviation of both abiotic and biotic stresses in wheat; (iv) stability and effectiveness are 2 major challenges during field application; (v) a STVAE pipeline that includes 5 processes, namely, Screen, Test, Validation, Application, and Evaluation, has been proposed for the capture and refinement of wheat-associated Bacillus spp. In particular, this review comprehensively addresses possible solutions, concerns, and criteria during the development of native Bacillus-based inoculants for sustainable wheat production.

Halotolerant Bacillus altitudinis WR10 improves salt tolerance in wheat via a multi-level mechanism.

Soil salinity is an important abiotic stress factor that seriously affects the crop growth and yield. Use of plant-derived microorganisms is a promising strategy to alleviate salt stress. In a previous study, the endophytic strain Bacillus altitudinis WR10 isolated from wheat roots showed high salt resistance. In this study, we investigated the efficacy of WR10 in improving the salt tolerance of wheat and its potential mechanisms using a hydroponic test. Under salt stress, WR10 inoculation significantly increased the lengths and dry weights of the roots and shoots, indicating that WR10 improves wheat salt tolerance at the seedling stage. WR10 inoculation significantly reduced Na+ accumulation and enhanced K+, P, and Ca2+ uptake in salt-stressed plants, which can be attributed to the upregulated gene expression of H+-ATPase as well as the P-solubilizing and biofilm-producing characteristics of WR10. At the transcriptional level, L-ascorbate peroxidase (APX), glutathione (GSH) synthetase related to GSH biosynthesis, and phenylpropanoid biosynthesis genes (CYP73A, 4CL, and CAD) were significantly upregulated, whereas those of GSH metabolism genes (glutathione S-transferase and gamma-glutamyltranspeptidase) were significantly downregulated in WR10-applied wheat roots under salt stress. These changes increased the APX activity and GSH levels and resulted in a decrease in hydrogen peroxide levels. Additionally, a decrease in proline content was observed in WR10-inoculated plants under salt stress because of WR10-induced upregulation of proline dehydrogenase gene expression. These results provide supporting evidence that WR10 improves wheat salt tolerance via more than one mechanism and open a window of opportunity for WR10 application in salinized soil.

Genetically Engineered Methanotroph as a Platform for Bioaugmentation of Chemical Pesticide Contaminated Soil.

Bioaugmentation is a promising alternative in soil remediation. One challenge of bioaugmentation is that exogenous pollutant-degrading microbes added to soil cannot establish enough biomass to eliminate pollutants. Considering that methanotrophs have a growth advantage in the presence of methane, we hypothesize that genetically engineered methanotrophs could degrade contaminants efficiently in soil with methane. Here, methanotroph Methylomonas sp. LW13, herbicide bensulfuron-methyl (BSM), and two kinds of soil were chosen to confirm this hypothesis. The unmarked gene knock-in method was first developed for strain LW13. Then, BSM hydrolase encoding gene sulE was inserted into the chromosome of strain LW13, conferring it BSM-degrading ability. After inoculation, the cell amount of strain LW13-sulE in soil raised considerably (over 100 fold in 9 days) with methane provision; meanwhile, >90% of BSM in soil was degraded. This study provides a proof of the concept that genetically engineered methanotroph is a potential platform for soil remediation.

The Operon Encoding Hydrolytic Dehalogenation of 4-Chlorobenzoate Is Transcriptionally Regulated by the TetR-Type Repressor FcbR and Its Ligand 4-Chlorobenzoyl Coenzyme A.

The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme A (CoA)-activation-type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the Pfcb -fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the P fcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5'-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3') that consists of two sequence repeats containing four pseudopalindromic sequences (5'-TCNATNGA-3'). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.

Methylomonas rhizoryzae sp. nov., a type I methanotroph isolated from the rhizosphere soil of rice.

A gammaproteobacterial methanotroph, strain GJ1T, was isolated from a rhizosphere soil sample of rice in Nanjing, China. The cells were Gram-negative, motile rods with a single polar flagellum, and they contained type I intracytoplasmic membranes. The cells formed pink colonies. The strain possessed both the particulate methane monooxygenase enzyme (pMMO) and the soluble methane monooxygenase enzyme (sMMO). pxmABC, encoding a divergent methane monooxygenase (pXMO), and nifH, which encodes dinitrogenase reductase, were also present. Methane and methanol were utilized as sole carbon sources, while other carbon sources, including acetate, pyruvate, succinate, citrate, malate, glucose, urea, methylamine, ethanol and formate, could not be utilized by strain GJ1T. Cell grew optimally at 25-33 °C (range 16-37 °C), pH 6.0-8.0 (range 5.5-8.5) and 0-1.2% NaCl (no growth above 1.5% NaCl). Phylogenetic analyses based on the 16S rRNA gene, pmoA and nifH showed that the isolate belongs to the genus Methylomonas of the family Methylococcaceae within the class Gammaproteobacteria. The major quinone was determined to be MQ-8, and the major fatty acids were observed to be C16:1 and C14:0. The genome size of strain GJ1T is about 4.55 Mb, and the DNA G + C content of the strain was determined to be 53.67 mol% within the range of the genus Methylomonas (47-58 mol%) reported at present. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain GJ1T and Methylomonas koyamae Fw12E-YT among the genus Methylomonas were the highest, and they were only 74.66% and 21.40%, respectively. In consequence, results of phenotypic characterization and phylogenetic analyses support strain GJ1T as a novel species within the genus Methylomonas, namely, Methylomonas rhizoryzae sp. nov.. The type strain is GJ1T (= ACCC 61706).

pheS AG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium.

Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheS AG , which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheS AG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The P tac promoter and the ribosome-binding site region of mmoX were employed to control pheS AG , producing the pheS AG -3 CSM. M. buryatense 5GB1C harboring pheS AG -3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheS AG -3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.

Biodegradation of plastic monomer 2,6-dimethylphenol by Mycobacterium neoaurum B5-4.

2,6-Dimethylphenol (2,6-DMP), an important chemical intermediate and the monomer of plastic polyphenylene oxide, is widely used in chemical and plastics industry. However, the pollution problem of 2,6-DMP residues is becoming increasingly serious, which is harmful to some aquatic animals. Microbial degradation provided an effective approach to eliminate DMPs in nature, which is considered as a prospective way to remediate DMPs-contaminated environments. But the 2,6-DMP-degrading bacteria is not available and the molecular mechanism of 2,6-DMP degradation is unclear as well. Here, a 2,6-DMP-degrading bacterium named B5-4 was isolated and identified as Mycobacterium neoaurum. M. neoaurum B5-4 could utilize 2,6-DMP as the sole carbon source for growth. Furthermore, M. neoaurum B5-4 could degrade 2,6-DMP with concentrations ranging from 1 to 500 mg L-1. Six intermediate metabolites of 2,6-DMP were identified and a metabolic pathway of 2,6-DMP in M. neoaurum B5-4 was proposed, in which 2,6-DMP was initially converted to 2,6-dimethyl-hydroquinone and 2,6-dimethyl-3-hydroxy-hydroquinone by two consecutive hydroxylations at C-4 and γ position; 2,6-dimethyl-3-hydroxy-hydroquinone was then subjected to aromatic ring ortho-cleavage to produce 2,4-dimethyl-3-hydroxymuconic acid, which was further transformed to citraconate, and subsequently into TCA cycle. In addition, toxicity bioassay of 2,6-DMP in water using zebrafish indicates that 2,6-DMP is toxic to zebrafish and M. neoaurum B5-4 could effectively eliminate 2,6-DMP in water to protect zebrafish from 2,6-DMP-induced death. This work provides a potential strain for bioremediation of 2,6-DMP-contaminated environments and lays a foundation for elucidating the molecular mechanism and genetic determinants of 2,6-DMP degradation.

The substrate specificity of aniline dioxygenase is mainly determined by two of its components: glutamine synthetase-like enzyme and oxygenase.

The residues of aniline and its derivatives are serious environment pollutants. Aniline dioxygenase (AD) derived from aerobic bacteria catalyzes the conversion of aniline to catechol, which has potential use in the bioremediation of aromatic amines and biorefining process. AD contains four components: a glutamine synthetase (GS)-like enzyme, a glutamine amidotransferase (GAT)-like enzyme, oxygenase, and reductase. ADs from diverse hosts exhibit different substrate specificities against aniline derivatives. However, what component of AD determines AD's substrate specificity is still unknown which limits the effects of extending AD's substrate spectrum through mutagenesis. Here, each component of two ADs (AtdA1A2A3A4A5 and AdoQTA1A2B) which have different substrate ranges was heterologously expressed and purified. The activity of both ADs was successfully constructed in vitro using the purified components. To identify the component that affects the substrate specificity of the ADs, the substrate specificity of each component was studied. The inability of AtdA1A2A3A4A5 to catalyze 4-methylaniline was determined with GS-like enzyme AtdA1; its inability to convert 2-isopropylaniline was caused by the oxygenase component, and its inability to convert 4-isopropylaniline was caused by both GS-like enzyme AtdA1 and oxygenase components. The inability of AdoQTA1A2B to catalyze 2-methylaniline was determined by GS-like enzyme AdoQ; its inability to convert 2-isopropylaniline was caused by both GS-like enzyme AdoQ and oxygenase components. Together, these results show that GS-like enzyme and oxygenase but not GAT-like enzyme or reductase play dominant roles in the substrate specificity of AD, and this finding will facilitate the engineering of AD to expand its substrate range.

Loneliness, Stress, and Depressive Symptoms Among the Chinese Rural Empty Nest Elderly: A Moderated Mediation Analysis.

This study aimed to explore the relationship of loneliness, perceived stress, and depressive symptoms among the Chinese rural empty nest elderly. The role of sense of coherence (SOC) as a moderating variable was investigated in a representative sample of older adults in Henan, China, from November 2016 to February 2017. Results indicated that perceived stress mediated the relationship between loneliness and depressive symptoms, and SOC acted as a moderator on this mediating model. The mediation effect of perceived stress was significant only when SOC was lower. The results emphasized the importance of perceived stress in older adults. SOC was a protective factor with regard to depressive symptoms, and improving SOC should be a focus of health promotion with the elderly.

Poor sleep quality and late-life depression among the elderly in urban communities in liaoning, China: A moderated mediation analysis.

This study aims to investigate a moderated mediation model in the relationship between poor sleep quality, depressive symptoms, perceived stress, sense of coherence. A cross-sectional survey was conducted from May to July 2015 in Liaoning Province, China. 1050 community residents aged ≥60 years were ultimately chosen to participate. The Chinese version of the Pittsburgh Sleep Quality Index (CPSQI), the Epidemiological Studies Depression Scale(CES-D),the Chinese version Perceived Stress Scale(CPSS), and the Sense of Coherence Scale (SOC) were used to estimate sleep quality, late-life depression, perceived stress, and sense of coherence (SOC) respectively. Pearson's correlations and the PROCESS macro were applied to analyze the data. The results showed that perceived stress partially mediated the association between poor sleep quality and depression symptoms (35% proportion mediated). In addition, the effect of poor sleep quality and the effect of perceived stress on late-life depression was moderated by SOC.The current findings indicates that the impact of poor sleep quality on late-life depression through perceived stress can be buffered when the elderly individuals have a high SOC. Interventions should therefore target individuals who are classified as poor sleepers but only have a lower SOC score. However, longitudinal studies should be conducted in future to substantiate these findings. Importantly, this research provides a useful theoretical and methodological approach for prevention of depression among the elderly in urban China.

The mediating role of depression on the relationship between housebound status and cognitive function among the elderly in rural communities: A cross-sectional study.

OBJECTIVE: To explore the relationship among housebound status, depression and cognitive function, and further to study whether depression can take on a mediator among elders in rural communities. METHODS: A cross-sectional survey was conducted among 720 community residents aged ≥60 years from March to May in 2016 in Hebei province, China. We used the Chinese version of Housebound scale, the Rasch-Derived of the Center for Epidemiological studies Depression scale (CES-D-R), and the Minimental State Examination Scale (MMSE) to estimate housebound status, depression, and cognitive function separately. Correlation, multiple linear regression, and structural equation modeling was used to data analyses. RESULTS: 712 completely replied questionnaires were finally used in the data analyses among 720 questionnaires, which indicated that effective response rate was 98.9%. Results indicated that the scores of housebound status were positively correlated with the MMSE scores. Being housebound correlated negatively with MMSE scores. Housebound status and three dimensions of CES-D-R (i.e. negative affect, positive affect, and interpersonal problems) were deemed as significant predictors of cognitive function among rural elders. The effect of housebound status on cognitive function was fully mediated by depression. CONCLUSIONS: Not all subscales of CES-D-R can affect MMSE scores among rural elders. There may be full mediation effects of depression within the impact of housebound status on cognitive function, mainly through negative affect, positive affect and interpersonal problems. It indicates that preventing the elderly from housebound status could relieve the decline of cognitive function by the intermediary role of depression.

Locomotive function and quality of life among older people in Liaoning, China: Falls efficacy as mediator or moderator?

OBJECTIVES: This study aimed to examine the role of falls efficacy in the relationship between the locomotive function and quality of life. METHODS: From March to May in 2016, we conducted a cross-sectional survey among 830 community residents aged ≥60 years from Jinzhou,China.GLFS-25 (25-question Geriatric Locomotive Function Scale), FES-I(International edition of Falls Efficacy Scale),and SF-12 (12-item Short Form Health Survey) were used to estimate locomotive function, falls efficacy and quality of life, respectively. The higher score of GLFS-25, the worse the locomotive function. RESULTS: Median age was 68.69 years (ranges 60-88). Locomotive function, falls efficacy and quality of life all presented a linear relationship within each other, locomotive function score was negatively correlated with falls efficacy score (-0.461, P < 0.01). locomotive function score was negatively correlated with quality of life score (-0.523, P < 0.01).Falls efficacy score was positively correlated with quality of life score (0.415, P < 0.01).Falls efficacy exerted both a mediating and moderating role between locomotive function and quality of life, and the mediation effect accounted for 45.5% of the total effect. CONCLUSIONS: Poorer locomotive function was associated with poorer quality of life, and greater falls efficacy was associated with better quality of life. In addition, falls efficacy was demonstrated to be both a mediator and moderator variable in the linkage between locomotive function and quality of life. Aged care professional practitioners and our policy makers should strengthen the awareness of the psychological role of the elderly falls efficacy.

Psychometric Assessment of the Depressive Cognition Scale Among Older Chinese People.

The study aimed to translate the Depressive Cognition Scale (DCS) into Chinese and to test its reliability and validity among Chinese older people. Using a cross-sectional design, a demographic questionnaire and Chinese versions of the Depressive Cognition Scale (DCS-CHI) and the Center for Epidemiological Studies-Depression Scale (CES-D) were administered. The sample consisted of 1673 older people who were from communities and hospitals. The Cronbach's alpha (α) of DCS-CHI was 0.91, and the test-retest correlation coefficient was 0.91 (95% CI, 0.86-0.95, p<0.001). The Content Validity Index (CVI) was found to be good. Exploratory factor analysis (EFA) resulted in a single factor that explained 58.46% of the total variance and all 8 items had strong factor loadings ranging from 0.62 to 0.83; confirmatory factory analysis (CFA) indicated all measurements of the structural model exceeded the recommended criteria, and the single factor solution of DCS-CHI had a good fit (χ2/df=2.45, GFI=0.99, AGFI=0.97, CFI=0.99, TLI=0.99, RMSEA=0.04, RMR=0.01, PCLOSE=0.79). The strong correlation of 0.81 (p<0.01) between the DCS-CHI and CES-D suggested good concurrent validity. Specifying the CES-D as the criterion, the area under the receiver operator characteristic (ROC) curve of the DCS-CHI for the optimal cut-point was 0.941(95%CI:0.919-0.963, p=0.000), the sensitivity and the specificity were 84.7% and 90.7% respectively, suggesting good predictive validity. The findings support the reliability and validity of the DCS-CHI as a measure of depressive cognitions that typically proceed more serious depressive symptoms among Chinese older adults.

Artificial induction of genetic competence in Bacillus amyloliquefaciens isolates.

OBJECTIVES: To induce natural genetic competence in Bacillus amyloliquefaciens isolates through overexpression of the master regulator, ComK, from B. subtilis (ComK Bsu ). RESULTS: Plasmid pUBXC carrying the xylose-inducible comK expression cassette was constructed using plasmid pUB110 as a backbone. Plasmid pUBXC could be transferred from B. subtilis to B. amyloliquefaciens through plasmid pLS20-mediated biparental conjugation. After being induced by xylose, four B. amyloliquefaciens strains harbouring plasmid pUBXC developed genetic competence. Under optimal conditions, the transformation efficiencies of plasmid DNA ranged from 129 ± 20.6 to 1.7 ± 0.1 × 105 cfu (colony-forming units) per µg DNA, and the transformation efficiencies of PCR-assembled deletion constructs ranged from 3.2 ± 0.76 to 3.5 ± 0.42 × 104 cfu per µg DNA in the four tested strains. CONCLUSION: Artificial induction of genetic competence through overexpressing ComK Bsu in B. amyloliquefaciens completed the tasks of replicative plasmid delivery and gene knockout via direct transformation of PCR-generated deletion cassettes.

Molecular analysis of proanthocyanidins related to pigmentation in brown cotton fibre (Gossypium hirsutum L.).

The structural characteristics and component differences of proanthocyanidins in brown and white cotton fibres were identified by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. Proanthocyanidins in brown and white cotton fibres were found to contain mainly procyanidin (PC) and prodelphidin (PD) units with 2, 3-cis form (epigallocatechin and epicatechin). However, part of the proanthocyanidins in the white cotton fibres were modified by acylation and were constitutively different from the proanthocyanidins in brown cotton fibres. The relative amount of PD was similar to that of PC in white cotton fibres, while proanthocyanidins in brown cotton fibres consisted mainly of PD units with a relative ratio of 9:1. In brown cotton fibres, the proanthocyanidin monomeric composition was consistent with the expression profiles of proanthocyanidin synthase genes, suggesting that anthocyanidin reductase represented the major flow of the proanthocyanidin biosynthesis pathway. In addition, the structural characteristics and component differences of proanthocanidins in brown and white cotton fibres suggested that quinones, the oxidation products of proanthocyanidins, were the direct contributors to colour development in brown cotton fibre. This was demonstrated by vanillin-HCl staining and Borntrager's test. Collectively, these data demonstrated that the biosynthesis of proanthocyanidins is a crucial pigmentation process in brown cotton fibre, and that quinones may represent the main pigments contributing to formation of the the brown colour. This study revealed the molecular basis of pigmentation in brown cotton fibres, and provided important insights for genetic manipulation of pigment production in cotton fibres.