Analysis of latrine fecal odor release pattern and the deodorization with composited microbial agent.

As an important source of malodor, the odor gases emitted from public toilet significantly interfered the air quality of living surroundings, resulting in environmental problem which received little attention before. Thus, this paper explored the odor release pattern of latrine feces and deodorization effect with composited microbial agent in Chengdu, China. The odor release rules were investigated in sealed installations with a working volume of 9 L for 20 days. The odor units (OU), ammonia (NH3), hydrogen sulfide (H2S) and total volatile organic compounds (TVOC) were selected to assess the release of malodorous gases under different temperature and humidity, while the highest malodor release was observed under 45℃, with OU and TVOC concentration was 643.91 ± 2.49 and 7767.33 ± 33.50 mg/m3, respectively. Microbes with deodorization ability were screened and mixed into an agent, which composited of Bacillus amyloliquefaciens, Lactobacillus plantarum, Enterococcus faecalis and Pichia fermentans. The addition of microbial deodorant could significantly suppress the release of malodor gas during a 20-day trial, and the removal efficiency of NH3, H2S, TVOC and OU was 81.50 %, 38.31 %, 64.38 %, and 76.86 %, respectively. The analysis of microbial community structure showed that temperature was the main environmental factor driving the microbial variations in latrine feces, while Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes were the main bacteria phyla involved in the formation and emission of malodorous gases. However, after adding the deodorant, the abundance of Bacteroidetes, Proteobacteria and Actinobacteria were decreased, while the abundance of Firmicutes was increased. Furthermore, P. fermentans successfully colonized in fecal substrates and became the dominant fungus after deodorization. These results expanded the understanding of the odor release from latrine feces, and the composited microbial deodorant provided a valuable basis to the management of odor pollution.

Tumor-associated monocytes promote mesenchymal transformation through EGFR signaling in glioma.

The role of brain immune compartments in glioma evolution remains elusive. We profile immune cells in glioma microenvironment and the matched peripheral blood from 11 patients. Glioblastoma exhibits specific infiltration of blood-originated monocytes expressing epidermal growth factor receptor (EGFR) ligands EREG and AREG, coined as tumor-associated monocytes (TAMo). TAMo infiltration is mutually exclusive with EGFR alterations (p = 0.019), while co-occurring with mesenchymal subtype (p = 4.7 × 10-7) and marking worse prognosis (p = 0.004 and 0.032 in two cohorts). Evolutionary analysis of initial-recurrent glioma pairs and single-cell study of a multi-centric glioblastoma reveal association between elevated TAMo and glioma mesenchymal transformation. Further analyses identify FOSL2 as a TAMo master regulator and demonstrates that FOSL2-EREG/AREG-EGFR signaling axis promotes glioma invasion in vitro. Collectively, we identify TAMo in tumor microenvironment and reveal its driving role in activating EGFR signaling to shape glioma evolution.

Performance evaluation of H2S and NH3 removal by biological trickling filter reactors with various fillers under heterotrophic conditions.

A pilot-scale biological trickling filter (BTF) reactor (13.5 L) packed with different fillers (Pine bark, Cinder, Straw, and MBBR (mobile bed biofilm reactor) filler was employed to evaluate their removal performance of H2S and NH3 after heterotrophic bacterium addition, and some parameters, including different packing heights, empty bed residence time (EBRT), inlet titers, loading ratios, and restart trial, were investigated in this study. According to the experimental results, BTF filled with pine bark exhibited better removal efficiency than other reactors under a variety of conditions. The removal efficiency of H2S and NH3 reached to as high as 81.31 % and 91.72 %, respectively, with the loading range of 3.29-67.70 g/m3·h. Moreover, due to the addition of heterotrophic bacterium, the removal efficiency was enhanced and capable to eliminate majority of H2S and NH3 even though the packing height was reduced to 400 mm. After 15 days of idle, the BTF reactor was able to resume rapidly and execute deodorization with high efficiency. The degradation mechanism was further explored by a thorough examination of microbial species which degraded contaminants, as well as by functional prediction and correlation analyses. In a word, these results laid a foundation for the application of heterotrophic microorganisms in BTF, which could improve the removal efficiency of biological deodorization.

Microbiologically induced calcite precipitation for in situ stabilization of heavy metals contributes to land application of sewage sludge.

Microbiologically induced calcite precipitation (MICP) has shed new light on solving the problem of in situ stabilization of heavy metals (HMs) in sewage sludge before land disposal. In this study, we examined whether MICP treatment can be integrated into a sewage sludge anaerobic digestion-land application process. Our results showed that MICP treatment not only prevented the transfer of ionic-state Cd from the sludge to the supernatant (98.46 % immobilization efficiency) but also reduced the soluble exchangeable Pb and Cd fractions by up to 100 % and 48.54 % and increased the residual fractions by 22.54 % and 81.77 %, respectively. In addition, the analysis of the stability of HMs in MICP-treated sludge revealed maximum reductions of 100 % and 89.56 % for TCLP-extractable Pb and Cd, respectively. Three-dimensional fluorescence, scanning electron microscopy-energy-dispersive X-ray spectroscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy analyses confirmed the excellent performance of the ureolytic bacteria Sporosarcina ureilytica ML-2 in the sludge system. High-throughput sequencing showed that the relative abundance of Sporosarcina sp. reached 53.18 % in MICP-treated sludge, and the urease metabolism functional genes unit increased by a maximum of 239.3 %. The MICP technology may be a feasible method for permanently stabilizing HMs in sewage sludge before land disposal.

Nitrogen removal characteristics of efficient heterotrophic nitrification-aerobic denitrification bacterium and application in biological deodorization.

A heterotrophic nitrifying aerobic denitrifying (HN-AD) strain HY-1 with excellent capacity, identified as Paracoccus denitrificans, was isolated from activated sludge. HY-1 was capable of removing NH4+, NO2-, and NO3- with the corresponding rate of 17.33 mg-N L-1 h-1, 21.83 mg-N L-1 h-1, and 32.37 mg-N L-1 h-1, as well as the mixture of multiple nitrogen sources. Meanwhile, HY-1 could execute denitrification function under anaerobic conditions with a rate of 14.56 mg-N L-1 h-1. HY-1 required less energy investment, which exhibited average denitrification rate of 5.19 mg-N L-1 h-1 at carbon-nitrogen ratio was 1. After nitrification-denitrification metabolic pathway analysis, HY-1 was applied in a biological trickling filter reactor for compost deodorization. The results showed that adding of HY-1 greatly reduced the ionic concentration of NH4+ and NO3- in the circulating liquid without impairing the deodorization effect (NH3 removal rate＞98.07%). These findings extend the field of application of HN-AD and provide new insights for biological deodorization.

Mechanism of microbiologically induced calcite precipitation for cadmium mineralization.

Microbiologically induced calcite precipitation (MICP) technology shows potential for remediating heavy metal pollution; however, the underlying mechanism of heavy metal mineralization is not well-understood, limiting the application of this technology. In this study, we targeted Cd contamination (using 15:1, 25:1, and 50:1 Ca2+/Cd2+ molar ratios) and showed that the ureolytic bacteria Sporosarcina ureilytica ML-2 removed >99.7 % Cd2+ with a maximum fixation capacity of 75.61 mg-Cd/g-CaCO3 and maximum precipitation production capacity of 135.99 mg-CaCO3/mg-cells. Quantitative PCR analysis showed that Cd2+ inhibited the expression of urease genes (ureC, ureE, ureF, and ureG) by 70 % in the ML-2 strain. Additionally, the pseudo-first-order kinetics model (R2 = 0.9886), intraparticle diffusion model (R2 = 0.9972), and Temkin isotherm model (R2 = 0.9828) described the immobilization process of Cd2+ by bio calcite in MICP-Cd system. The three Cd2+ mineralization products generated by MICP were attributed to surface precipitation (Cd2+ → Cd(OH)2), direct binding with the CO32-/substitution calcium site of calcite (Cd2+ → CdCO3, otavite), and calcite lattice vacancy anchors (Cd2+ → (CaxCd1-x)CO3). Our findings improve the understanding of the mechanisms by which MICP can achieve in situ stabilization of heavy metals.

Alantolactone-Loaded Pegylated Prodrug Nanocarriers for Synergistic Treatment of Cisplatin-Resistant Ovarian Cancer via Reactivating Mitochondrial Apoptotic Pathway.

Ovarian cancer (OV) seriously damages women's health because of refractory OV and the development of platinum (Pt) resistance. New treatment strategies are urgently needed to deal with the treatment of cisplatin-resistant OV. Here, a reduction-sensitive pegylated Pt(IV) prodrug was synthesized by amidation of methoxy polyethylene glycol amine (PEG750-NH2) with monocarboxylic Pt(IV) prodrug (Pt(IV)-COOH). Then alantolactone (AL) loaded PEG-Pt(IV) nanocarriers (NP(Pt)@AL) were prepared. In the cisplatin-resistant model of OV, cancer cells actively ingest NP(Pt)@AL through endocytosis, and AL and Pt(II) were disintegrated and released under high intracellular reductant condition. The activity of thioredoxin reductase 1 (TrxR1) inhibited by AL and the adducts of Pt(II) with mitochondrial DNA (mDNA) can costimulate reactive oxygen species (ROS) and reactivate the mitochondrial pathway of apoptosis. Meanwhile, Pt(II) binds with nuclear DNA (nDNA) to jointly promote cell apoptosis. Both in vitro and in vivo results demonstrated that NP(Pt)@AL could effectively reverse the drug resistance and displayed excellent synergistic therapeutic efficacy on platinum-resistant OV with high safety. Therefore, reactivation of the mitochondrial pathway of apoptosis would be a potential strategy to improve the therapeutic effect of Pt-based chemotherapy and even reverse drug resistance.

Modular configurations of living biomaterials incorporating nano-based artificial mediators and synthetic biology to improve bioelectrocatalytic performance: A review.

Currently, the industrial application of bioelectrochemical systems (BESs) that are incubated with natural electrochemically active microbes (EABs) is limited due to inefficient extracellular electron transfer (EET) by natural EABs. Notably, recent studies have identified several novel living biomaterials comprising highly efficient electron transfer systems allowing unparalleled proficiency of energy conversion. Introduction of these biomaterials into BESs could fundamentally increase their utilization for a wide range of applications. This review provides a comprehensive assessment of recent advancements in the design of living biomaterials that can be exploited to enhance bioelectrocatalytic performance. Further, modular configurations of abiotic and biotic components promise a powerful enhancement through integration of nano-based artificial mediators and synthetic biology. Herein, recent advancements in BESs are synthesized and assessed, including heterojunctions between conductive nanomaterials and EABs, in-situ hybrid self-assembly of EABs and nano-sized semiconductors, cytoprotection in biohybrids, synthetic biological modifications of EABs and electroactive biofilms. Since living biomaterials comprise a broad range of disciplines, such as molecular biology, electrochemistry and material sciences, full integration of technological advances applied in an interdisciplinary framework will greatly enhance/advance the utility and novelty of BESs. Overall, emerging fundamental knowledge concerning living biomaterials provides a powerful opportunity to markedly boost EET efficiency and facilitate the industrial application of BESs to meet global sustainability challenges/goals.

Study on anaerobic co-digestion of municipal sewage sludge and fruit and vegetable wastes: Methane production, microbial community and three-dimension fluorescence excitation-emission matrix analysis.

Constantly increased sewage sludge (SS) and fruit and vegetable wastes (FVW) are becoming the major organic solid wastes in human society. Thus, anaerobic digestion is employed as a low carbon energy strategy to reduce their environmental pollution risk. Anaerobic co-digestion system was developed based on the carbon to nitrogen ratio strategy. Results showed that the daily biogas production was higher in co-digester, and the volumetric biogas production rate (VBPR) significantly enhanced for 1.3 âˆ¼ 3 folds, and the highest VBPR was 2.04 L/L • day with optimal OLR of 2.083 Kg L-1 d-1. Analytic results indicated that co-digestion could improve the biodegradable of feedstocks, which transforming to more VFAs and biogas. Compared with mono SS digester, mixed substrates relieved ammonia nitrogen inhibition and enhanced the hydrolytic acidification and methanogenesis. Meanwhile, the excessive humification of organics was suppressed. This study supported the concepts of improving carbon recovery from SS and FVW.

Improvement of sewage sludge anaerobic digestion through synergistic effect combined trace elements enhancer with enzyme pretreatment and microbial community response.

In this study, a double E strategy (enzymes and enhancer) characterized by high efficiency for enhancing sewage sludge anaerobic digestion (AD) is proposed. This strategy combines addition of trace elements (TEs) enhancer and enzyme pretreatment, inducing a synergistic effect on AD, and it is more effective and economical compared with TEs addition or enzyme pretreatment in isolation. When adding 400 U/g cocktail enzymes and 1.24% trance elements enhancers, the cumulative methane production and the maximum daily methane increased yield by 45.29% and 84.7%, respectively. According to microbial community analysis, the double E strategy significantly motivate the growth of acetogens and protein fermenting bacterium. The relative abundance of Fermentimonas and Lutispora increased by 6.15% and 5.4%, respectively. Archaeal community analysis and changes in the mcrA gene abundance demonstrate enrichment of hydrogenotrophic methanogens, with the methanogens exhibiting high vitalities and stress resistance. The double E strategy could be a promising way to improve industrial sewage sludge AD efficiency.

Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.

Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 µg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Role of RelA and SpoT on ppGpp Production, Stress Response, Growth Regulation, and Pathogenicity in Xanthomonas campestris pv. campestris.

The alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen. IMPORTANCE Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria.

Microbiologically induced calcite precipitation technology for mineralizing lead and cadmium in landfill leachate.

As a new bioremediation technology for toxic metals, microbiologically induced calcite precipitation (MICP) is gradually becoming a research focus. This study investigated the application of MICP to mineralize toxic metals (lead and cadmium) in landfill leachate for the first time. In the experiment of remediating synthetic landfill leachate (SLL) contaminated by Pb2+, 100% of the 20 mg/L Pb2+ was removed when the maximum urease activity was only 20.96 U/ml. Scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS) and laser particle size characterizations of the precipitates indicate the formation of agglomerated square particles, 76.9% of which had sizes that ranged from 33.93 to 57.06 µm. Fourier transform infrared spectroscopic and X-ray diffraction analyses confirmed that the precipitates consisted predominantly of calcite crystals, and the unit cell lattice constants of the precipitates (a = b = 4.984 Å, c = 17.171 Å) matched those of calcite, while lead was fixed as hydrocerussite. In addition, the Pb-MICP precipitates were stable under continuous acid degradation (pH = 5.5), and only 1.76% of the lead was released after 15 days. In the verification test of toxic metals remediation in a real landfill leachate (RLL), all of the Pb2+ and Cd2+ (initial concentrations: Pb2+ = 25 mg/L; Cd2+ = 5.6205 mg/L) was mineralized simultaneously, which further confirmed the feasibility of MICP for toxic metal remediation in landfill leachate. However, optimizing the urea dosage and combining the ammonium recovery are necessary strategies required for improving the economic and environmental benefits of the MICP process.

Analyses of deodorization performance of mixotrophic biotrickling filter reactor using different industrial and agricultural wastes as packing material.

In this study, to efficiently remove malodorous gas and reduce secondary pollution under mixotrophic conditions, pine bark, coal cinder, straw and mobile bed biofilm reactor (MBBR) fillers were used as packing materials in a biological trickling filter (BTF) to simultaneously treat high-concentration H2S and NH3. The results showed that the removal rate of BTF-A filled with pine bark was the highest, which was 86.31% and 94.06% under the H2S and NH3 loading rates of 53.59 g/m³·h while the empty bed residence time (EBRT) was 40.5 s. The theoretical maximum load was obtained by fitting the kinetic curve, and the value were 90.09 g H2S m-³·h-1 and 172.41 g NH3 m-³·h-1. Meanwhile, after treating with 720 ppm of NH3, the average concentration of NO3- in the BTF circulating fluid was only 127.58 mg/L, indicating the better performance of secondary pollutants control. Microbiological analysis showed that Dokdonella, Micropruina, Candidatus_Alysiosphaera, Nakamurella and Thiobacillus possessed high abundance at the genus level, and their entire percentage in four BTF reactors were 62.87%, 46.32%, 47.98%, and 57.35% respectively. It is worthwhile that the genera Comamonas and Trichococcus with heterotrophic nitrification and aerobic denitrification capabilities and proportion of 3.66%, 1.45%, 5.43%, and 3.23% were observed in four reactors.

Poly-γ-glutamic acid production by simultaneous saccharification and fermentation using corn straw and its fertilizer synergistic effect evaluation.

Agricultural wastes rich in lignocellulosic biomass have been used in the production of poly-γ-glutamic acid (γ-PGA) through separate hydrolysis and fermentation (SHF), but this process is complicated and generates a lot of wastes. In order to find a simpler and greener way to produce γ-PGA using agricultural wastes, this study attempted to establish simultaneous saccharification and fermentation (SSF) with citric acid-pretreated corn straw. The possibility of Bacillus amyloliquefaciens JX-6 using corn straw as substrate to synthesize γ-PGA was validated, and the results showed that increasing the proportion of glucose in the substrate could improve the γ-PGA yield. Based on these preliminary results, the corn straw was pretreated using citric acid. Then, the liquid fraction (xylan-rich) was used for cultivation of seed culture, and the solid fraction (glucan-rich) was used as the substrate for SSF. In a 10-L fermenter, the maximum cumulative γ-PGA concentration in batch and fed-batch SSF were 5.08 ± 0.78 g/L and 10.78 ± 0.32 g/L, respectively. Moreover, the product from SSF without γ-PGA extraction was used as a fertilizer synergist, increasing the yield of pepper by 13.46% (P < 0.05). Our study greatly simplified the production steps of γ-PGA, and each step achieved zero emission as far as possible. The SSF process for γ-PGA production provided a simple and green way for lignocellulose biorefinery and sustainable cultivation in agriculture.

Extraction and detection of guanosine 5'-diphosphate-3'-diphosphate in amino acid starvation cells of Clavibacter michiganensis.

Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the stringent response. This study reported the extraction of ppGpp from the Gram-positive bacteria Clavibacter michiganensis through methods using formic acid, lysozyme, or methanol. Following extraction, ppGpp was detected through ultra-high-performance liquid chromatography (UHPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methanol method showed the highest extraction efficiency for ppGpp among the three methods tested. C. michiganensis cells in exponential growth phase was induced in amino acid starvation by serine hydroxamate (SHX) and used for ppGpp extraction and detection. When using the methanol extraction method, the results showed that ppGpp concentrations in SHX-treated samples were 15.645 nM, 17.656 nM, 20.372 nM, and 19.280 nM at 0 min, 15 min, 30 min and 1 h, respectively, when detected using LC-MS/MS. This is the first report on ppGpp extraction and detection in Clavibacter providing a new idea and approach for nucleotide detection and extraction in bacteria.

Role of Penicillin-Binding Proteins in the Viability, Morphology, Stress Tolerance, and Pathogenicity of Clavibacter michiganensis.

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of the viable but nonculturable (VBNC) state, which allows non-spore-forming bacteria to survive adverse environmental conditions. The current study found that Clavibacter michiganensis has seven PBPs. Mutant analysis indicated that deletion of either of the class B PBPs was lethal and that the class A PBPs had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having high sensitivity to osmotic stress and lysozyme treatment and surprisingly high pathogenicity. The double mutant, ΔdacB/ΔpbpE, also had a slightly altered phenotype, resulting in longer cells. Further analysis revealed that both mutants had high sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed that the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology and has serious implications for disease management.

iTRAQ-based proteomic analyses of the plant-pathogenic bacterium Acidovorax citrulli during entrance into and resuscitation from the viable but nonculturable state.

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) disease, infects cucurbit crops including watermelon and melon. This bacterium can enter the viable but nonculturable (VBNC) state following exposure to copper sulfate. Moreover, copper-induced VBNC A. citrulli cells can be resuscitated by EDTA. In this study, isobaric tag for relative and absolute quantification (iTRAQ) was used to compare protein profiles of VBNC cells, resuscitated cells at different stages and log-phase cells of the A. citrulli model strain AAC00-1. A total of 2672 proteins were identified, with 60 being differentially abundant in VBNC cells compared with log-phase cells, and 469 being differentially abundant in resuscitated cells compared with VBNC cells. Proteins involved in the arginine and proline metabolism pathway and degradation of aromatic compounds could be important for the VBNC cells. In the early resuscitation process, proteins associated with carbon metabolism, and degradation of naphthalene and aromatic compounds were significantly enriched, while proteins involved in oxidative phosphorylation, bacterial chemotaxis, ABC transporters and quorum sensing were significantly enriched at the late resuscitation stages. This is the first study reporting thorough protein profile analyses of VBNC and resuscitating cells of a plant-pathogenic bacterium. BIOLOGICAL SIGNIFICANCE: The VBNC state is a dormant-like condition that was reported to occur in many bacterial species, upon facing a variety of environmental stresses. Acidovorax citrulli is a seed borne pathogenic bacterium that threatens cucurbit production worldwide. Moreover, A. citrulli can enter into the VBNC state after treatment of copper sulfate, thus increasing its survival and dissemination probabilities. This study enriches our understanding of the mechanisms of entrance into and resuscitation from the VBNC state of this important plant-pathogenic bacterium. This knowledge could be exploited in the future to develop novel approaches to interfere with these processes, thus contributing to a more efficient management of this pathogen. In a broader perspective, the knowledge emerging from this study has implications to the general understanding of the VBNC state in bacteria.

Evaluation of suitable reference genes for normalization of quantitative reverse transcription PCR analyses in Clavibacter michiganensis.

Clavibacter michiganensis, the causal agent of bacterial canker of tomato, is a Gram-positive bacterium and a model for studying plant diseases. The real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay is widely used to quantify gene expression in plant pathogenic bacteria. However, accurate quantification of gene expression requires stably expressed reference genes that are consistently expressed during the experimental conditions of interest. The use of inappropriate reference genes leads to a misinterpretation of gene expression data and false conclusions. In current study, we empirically assessed the expression stability of six housekeeping genes (gyrB, rpoB, tufA, bipA, gapA, and pbpA) of C. michiganensis under five experimental conditions using two algorithms, geNorm and NormFinder. C. michiganensis expressed gyrB, bipA, and gapA stably when growing in nutrient-rich broth (TBY broth and modified M9 broth). We concluded that pbpA, tufA, and gyrB were suitable reference genes in C. michiganensis-tomato interaction studies. We also recommended bipA and rpoB to be used to study bacterial gene expression under nutrient-poor conditions. Finally, gyrB, pbpA, and rpoB can be used to normalize the quantification of C. michiganensis gene expression while the bacterium is in the viable but nonculturable (VBNC) state. This study identified the most suitable reference genes depending on the experimental conditions for calibrating real-time qRT-PCR analyses of C. michiganensis and will be useful in studies that seek to understand the molecular interactions between C. michiganensis and tomato.