Prediction of response to neoadjuvant chemo-immunotherapy in patients with esophageal squamous cell carcinoma by a rapid breath test.

BACKGROUND: Neoadjuvant chemo-immunotherapy combination has shown remarkable advances in the management of esophageal squamous cell carcinoma (ESCC). However, the identification of a reliable biomarker for predicting the response to this chemo-immunotherapy regimen remains elusive. While computed tomography (CT) is widely utilized for response evaluation, its inherent limitations in terms of accuracy are well recognized. Therefore, in this study, we present a novel technique to predict the response of ESCC patients before receiving chemo-immunotherapy by testing volatile organic compounds (VOCs) in exhaled breath. METHODS: This study employed a prospective-specimen-collection, retrospective-blinded-evaluation design. Patients' baseline breath samples were collected and analyzed using high-pressure photon ionization time-of-flight mass spectrometry (HPPI-TOFMS). Subsequently, patients were categorized as responders or non-responders based on the evaluation of therapeutic response using pathology (for patients who underwent surgery) or CT images (for patients who did not receive surgery). RESULTS: A total of 133 patients were included in this study, with 91 responders who achieved either a complete response (CR) or a partial response (PR), and 42 non-responders who had stable disease (SD) or progressive disease (PD). Among 83 participants who underwent both evaluations with CT and pathology, the paired t-test revealed significant differences between the two methods (p < 0.05). For the breath test prediction model using breath test data from all participants, the validation set demonstrated mean area under the curve (AUC) of 0.86 ± 0.06. For 83 patients with pathological reports, the breath test achieved mean AUC of 0.845 ± 0.123. CONCLUSIONS: Since CT has inherent weakness in hollow organ assessment and no other ideal biomarker has been found, our study provided a noninvasive, feasible, and inexpensive tool that could precisely predict ESCC patients' response to neoadjuvant chemo-immunotherapy combination using breath test based on HPPI-TOFMS.

Multi-omics analysis reveals NNMT as a master metabolic regulator of metastasis in esophageal squamous cell carcinoma.

Metabolic reprogramming has been observed in cancer metastasis, whereas metabolic changes required for malignant cells during lymph node metastasis of esophageal squamous cell carcinoma (ESCC) are still poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of paired ESCC tumor tissues and lymph nodes to uncover the reprogramming of tumor microenvironment (TME) and metabolic pathways. By integrating analyses of scRNA-seq data with metabolomics of ESCC tumor tissues and plasma samples, we found nicotinate and nicotinamide metabolism pathway was dysregulated in ESCC patients with lymph node metastasis (LN+), exhibiting as significantly increased 1-methylnicotinamide (MNA) in both tumors and plasma. Further data indicated high expression of N-methyltransferase (NNMT), which converts active methyl groups from the universal methyl donor, S-adenosylmethionine (SAM), to stable MNA, contributed to the increased MNA in LN+ ESCC. NNMT promotes epithelial-mesenchymal transition (EMT) and metastasis of ESCC in vitro and in vivo by inhibiting E-cadherin expression. Mechanically, high NNMT expression consumed too much active methyl group and decreased H3K4me3 modification at E-cadherin promoter and inhibited m6A modification of E-cadherin mRNA, therefore inhibiting E-cadherin expression at both transcriptional and post-transcriptional level. Finally, a detection method of lymph node metastasis was build based on the dysregulated metabolites, which showed good performance among ESCC patients. For lymph node metastasis of ESCC, this work supports NNMT is a master regulator of the cross-talk between cellular metabolism and epigenetic modifications, which may be a therapeutic target.

Characteristics and Risk Factors for Persistent Cough After Pulmonary Resection.

BACKGROUND: Persistent cough after pulmonary resection may reduce quality of life for patients. However, there remains a lack of description of clinical characteristics and the risk factors for persistent cough after pulmonary resection. This study aimed to describe the characteristics of persistent cough after pulmonary resection and investigate independent risk factors for it. METHODS: This single-institution study retrospectively included 901 consecutive patients who had undergone thoracoscopic pulmonary resection between June 2019 and December 2020. The characteristics of persistent cough after pulmonary resection are described, and univariable and multivariable regression analyses were performed to identify the independent risk factors for persistent cough after pulmonary resection. RESULTS: Persistent cough after pulmonary resection occurred in 190 (21.1%) of the patients. It was usually an irritating dry cough (75.3%) that appeared on postoperative day 7 (interquartile range [IQR], 6-9) and lasted for approximately 5 (IQR, 2-6) months. It was often induced by a pungent smell, cold air, deep inhalation, speaking, postural changes, pungent food, or emotional excitement. Multivariable analyses showed that resection of the right upper lobe (odds ratio [OR] 2.311, 95% CI 1.246-4.285) and mediastinal lymph node removal (OR 3.686, 95% CI 2.140-6.346) were independently associated with the risk of persistent cough after pulmonary resection. CONCLUSIONS: Persistent cough after pulmonary resection is a common complication that should receive more attention. Mediastinal lymph node removal and resection of the right upper lobe may be independent risk factors for persistent cough after pulmonary resection.

Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in Paenibacillus sabinae T27.

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH4+) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Zinc-Responsive Regulator Zur Regulates Zinc Homeostasis, Secondary Metabolism, and Morphological Differentiation in Streptomyces avermitilis.

Zinc is an essential cofactor for many metal enzymes and transcription regulators. Zn2+ availability has long been known to affect antibiotic production and morphological differentiation of Streptomyces species. However, the molecular mechanism whereby zinc regulates these processes remains unclear. We investigated the regulatory roles of the zinc-sensing regulator Zur in Streptomyces avermitilis. Our findings demonstrate that Zur plays an essential role in maintaining zinc homeostasis by repressing the expression of the zinc uptake system ZnuACB and alternative non-zinc-binding ribosomal proteins and promoting the expression of zinc exporter ZitB. Deletion of the zur gene resulted in decreased production of avermectin and oligomycin and delayed morphological differentiation, and these parameters were restored close to wild-type levels in a zur-complemented strain. Zur bound specifically to Zur box in the promoter regions of avermectin pathway-specific activator gene aveR, oligomycin polyketide synthase gene olmA1, and filipin biosynthetic pathway-specific regulatory genes pteR and pteF. Analyses by reverse transcription quantitative PCR and luciferase reporter systems indicated that Zur directly activates the transcription of these genes, i.e., that Zur directly activates biosynthesis of avermectin and oligomycin. Zur positively regulated morphological development by repressing the transcription of differentiation-related genes ssgB and minD2. Our findings, taken together, demonstrate that Zur in S. avermitilis directly controls zinc homeostasis, biosynthesis of avermectin and oligomycin, and morphological differentiation. IMPORTANCE Biosynthesis of secondary metabolites and morphological differentiation in bacteria are affected by environmental signals. The molecular mechanisms whereby zinc availability affects secondary metabolism and morphological differentiation remain poorly understood. We identified several new target genes of the zinc response regulator Zur in Streptomyces avermitilis, the industrial producer of avermectin. Zur was found to directly and positively control avermectin production, oligomycin production, and morphological differentiation in response to extracellular Zn2+ levels. Our findings clarify the regulatory functions of Zur in Streptomyces, which involve linking environmental Zn2+ status with control of antibiotic biosynthetic pathways and morphological differentiation.

Avermectin B1a production in Streptomyces avermitilis is enhanced by engineering aveC and precursor supply genes.

Avermectins (AVEs) are economically potent anthelmintic agents produced by Streptomyces avermitilis. Among eight AVE components, B1a exhibits the highest insecticidal activity. The purpose of this study was to enhance B1a production, particularly in the high-yielding industrial strain A229, by a combination strategy involving the following steps. (i) aveC gene was engineered to increase B1a:B2a ratio. Three aveC variants (aveC2m, aveC5m, and aveC8m, respectively encoding two, five, and eight amino acid mutations) were synthesized by fusion PCR. B1a:B2a ratio in A229 derivative having kasOp*-controlled aveC8m reached 1.33 (B1a and B2a titers were 8120 and 6124 µg/mL). Corresponding values in A229 were 0.99 and 6447 and 6480 µg/mL. (ii) ß-oxidation pathway genes fadD and fadAB were overexpressed in wild-type (WT) strain and A229 to increase supply of acyl-CoA precursors for AVE production. The resulting strains all showed increased B1a titer. Co-overexpression of pkn5p-driven fadD and fadAB in A229 led to B1a titer of 8537 µg/mL. (iii) Genes bicA and ecaA involved in cyanobacterial CO2-concentrating mechanism (CCM) were introduced into WT and A229 to enhance carboxylation velocity of acetyl-CoA and propionyl-CoA carboxylases, leading to increased supply of malonyl- and methylmalonyl-CoA precursors and increased B1a titer. Co-expression of bicA and ecaA in A229 led to B1a titer of 8083 µg/mL. (iv) aveC8m, fadD-fadAB, and bicA-ecaA were co-overexpressed in A229, resulting in maximal B1a titer (9613 µg/mL; 49.1% increase relative to A229). Our findings demonstrate that the combination strategy we provided here is an efficient approach for improving B1a production in industrial strains.Key points• aveC mutation increased avermectin B1a:B2a ratio and B1a titer.• Higher levels of acyl-CoA precursors contributed to enhanced B1a production.• B1a titer in an industrial strain was increased by 49.1% via a combination strategy.

Heat Shock Repressor HspR Directly Controls Avermectin Production, Morphological Development, and H2O2 Stress Response in Streptomyces avermitilis.

The heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces, which accounts for ∼70% of commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H2O2 stress responses in the industrially important species Streptomyces avermitilis. HspR directly activated ave structural genes (aveA1 and aveA2) and H2O2 stress-related genes (katA1, catR, katA3, oxyR, ahpC, and ahpD), whereas it directly repressed heat shock genes (HSGs) (the dnaK1-grpE1-dnaJ1-hspR operon, clpB1p, clpB2p, and lonAp) and developmental genes (wblB, ssgY, and ftsH). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to corepress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs (htpG, hsp18_1, and hsp18_2) different from those of HspR (clpB1p, clpB2p, and lonAp). A consensus HspR-binding site, 5'-TTGANBBNNHNNNDSTSHN-3', was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in the coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H2O2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors but not as activators. The results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP cross talks with HspR at dnaK1p to coregulate the heat shock response (HSR), but it also has its own specific target heat shock genes (HSGs). The novel role of PhoP in the HSR further demonstrates the importance of this regulator in Streptomyces. Overexpression of hspR strongly enhanced avermectin production in Streptomyces avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP and facilitate methods for antibiotic overproduction in Streptomyces species.

SufR, a [4Fe-4S] Cluster-Containing Transcription Factor, Represses the sufRBDCSU Operon in Streptomyces avermitilis Iron-Sulfur Cluster Assembly.

Iron-sulfur (Fe-S) clusters are ubiquitous and versatile inorganic cofactors that are crucial for many fundamental bioprocesses in nearly all organisms. How cells maintain Fe-S cluster homeostasis is not well understood in Gram-positive bacteria. Genomic analysis showed that the Suf system, which is encoded by the sufRBDCSU operon, is the sole Fe-S cluster assembly system in the genus StreptomycesStreptomyces avermitilis is the industrial producer of avermectins, which are widely used as agricultural pesticides and antiparasitic agents. sufR (SAV6324) encodes a putative ArsR-family transcriptional regulator, which was characterized as a repressor of the sufRBDCSU operon in this investigation. Spectroscopy and mass spectrometry demonstrated that anaerobically isolated SufR contained an oxidation-sensitive [4Fe-4S] cluster and existed as a homodimer. Electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analyses revealed that [4Fe-4S]-SufR bound specifically and tightly to a 14-bp palindromic sequence (CAAC-N6-GTTG) in the promoter region of the sufR operon, repressing expression of the sufRBDCSU operon. The presence of the [4Fe-4S] cluster is critical for the DNA-binding activity of SufR. Cys182, Cys195, and Cys223 in the C-terminal region of SufR are essential for [4Fe-4S] cluster coordination, but Cys178 is not. The fourth non-Cys ligand in coordination of the [4Fe-4S] cluster for SufR remains to be identified. The findings clarify the transcriptional control of the suf operon by [4Fe-4S] SufR to satisfy the various Fe-S cluster demands. SufR senses the intracellular Fe-S cluster status and modulates the expression of the sole Fe-S cluster assembly system via its Fe-S cluster occupancy.IMPORTANCE Fe-S clusters function as cofactors of proteins controlling diverse biological processes, such as respiration, photosynthesis, nitrogen fixation, DNA replication, and gene regulation. The mechanism of how Actinobacteria regulate the expression of the sole Fe-S cluster assembly system in response to the various Fe-S cluster demands remains to be elucidated. In this study, we showed that SufR functions as a transcriptional repressor of the sole Fe-S cluster assembly system in the avermectin producer S. avermitilis [4Fe-4S]-SufR binds to the promoter region of the suf operon and represses its expression. When Fe-S cluster levels are insufficient, SufR loses its [4Fe-4S] cluster and DNA-binding activity. Apo-SufR dissociates from the promoter region of suf operon, and the expression of the suf system is strongly increased by derepression to promote the synthesis of Fe-S clusters. The study clarifies how Streptomyces maintains its Fe-S cluster homeostasis through the activity of SufR to modulate the various Fe-S cluster demands.

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression, antibiotic biosynthesis, and morphological development in Streptomyces avermitilis.

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5'-TTKAMKHSTTSAV-3', was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.

AccR, a TetR Family Transcriptional Repressor, Coordinates Short-Chain Acyl Coenzyme A Homeostasis in Streptomyces avermitilis.

Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are the most common extender units for the biosynthesis of fatty acids and polyketides in Streptomyces, an industrially important producer of polyketides. Carboxylation of acetyl- and propionyl-CoAs is an essential source of malonyl- and methylmalonyl-CoAs; therefore, acyl-CoA carboxylases (ACCases) play key roles in primary and secondary metabolism. The regulation of the expression of ACCases in Streptomyces spp. has not been investigated previously. We characterized a TetR family transcriptional repressor, AccR, that mediates intracellular acetyl-, propionyl-, methylcrotonyl-, malonyl-, and methylmalonyl-CoA levels by controlling the transcription of genes that encode the main ACCase and enzymes associated with branched-chain amino acid metabolism in S. avermitilis AccR bound to a 16-nucleotide palindromic binding motif (GTTAA-N6-TTAAC) in promoter regions and repressed the transcription of the accD1A1-hmgL-fadE4 operon, echA8, echA9, and fadE2, which are involved in the production and assimilation of acetyl- and propionyl-CoAs. Methylcrotonyl-, propionyl-, and acetyl-CoAs acted as effectors to release AccR from its target DNA, resulting in enhanced transcription of target genes by derepression. The affinity of methylcrotonyl- and propionyl-CoAs to AccR was stronger than that of acetyl-CoA. Deletion of accR resulted in increased concentrations of short-chain acyl-CoAs (acetyl-, propionyl-, malonyl-, and methylmalonyl-CoAs), leading to enhanced avermectin production. Avermectin production was increased by 14.5% in an accR deletion mutant of the industrial high-yield strain S. avermitilis A8. Our findings clarify the regulatory mechanisms that maintain the homeostasis of short-chain acyl-CoAs in StreptomycesIMPORTANCE Acyl-CoA carboxylases play key roles in primary and secondary metabolism. However, the regulation of ACCase genes transcription in Streptomyces spp. remains unclear. Here, we demonstrated that AccR responded to intracellular acetyl-, propionyl-, and methylcrotonyl-CoA availability and mediated transcription of the genes related to production and assimilation of these compounds in S. avermitilis When intracellular concentrations of these compounds are low, AccR binds to target genes and represses their transcription, resulting in low production of malonyl- and methylmalonyl-CoAs. When intracellular acetyl-, propionyl-, and methylcrotonyl-CoA concentrations are high, these compounds bind to AccR to dissociate AccR from target DNA, promoting the conversion of these compounds to malonyl- and methylmalonyl-CoAs. This investigation revealed how AccR coordinates short-chain acyl-CoA homeostasis in Streptomyces.

BldD, a master developmental repressor, activates antibiotic production in two Streptomyces species.

BldD generally functions as a repressor controlling morphological development of Streptomyces. In this work, evidences that BldD also activates antibiotic production are provided. In Streptomyces roseosporus (which produces daptomycin widely used for treatment of human infections), deletion of bldD notably reduced daptomycin production, but enhanced sporulation. BldD stimulated daptomycin production by directly activating transcription of dpt structural genes and dptR3 (which encodes an indirect activator of daptomycin production), and repressed its own gene. BldD-binding sites on promoter regions of dptE, dptR3, and bldD were all found to contain BldD box-like sequences, facilitating prediction of new BldD targets. Two Streptomyces global regulatory genes, adpA and afsR, were confirmed to be directly activated by BldD. The protein AfsR was shown to act as an activator of daptomycin production, but a repressor of development. BldD directly represses nine key developmental genes. In Streptomyces avermitilis (which produces effective anthelmintic agents avermectins), BldD homolog (BldDsav) directly activates avermectin production through ave structural genes and cluster-situated activator gene aveR. This is the first report that BldD activates antibiotic biosynthesis both directly and via a cascade mechanism. BldD homologs are widely distributed among Streptomyces, our findings suggest that BldD may activate antibiotic production in other Streptomyces species.

Epsilon-Fe2O3 is a novel intermediate for magnetite biosynthesis in magnetotactic bacteria.

BACKGROUND: Natural biological magnetite nanoparticles are widely distributed from microorganisms to humans. It is found to be very important in organisms, especially in navigation. Moreover, purified magnetite nanoparticles also have potential applications in bioengineering and biomedicine. Magnetotactic bacteria (MTB) is considered one of the most abundant species around the world which can form intracellular membrane enveloped magnetic nanoparticles, referred to as magnetosomes. To our knowledge, the biomineralization of magnetosome in MTB involves a serious of genes located on a large unstable genomic region named magnetosome island, which specially exists in MTB. The magnetite core of magnetosome formed via a Fe (III) ion intermediates, for instance, α-Fe2O3 and ferrihydrite. Though the biosynthesis of magnetosome represents a general biomineralization mechanism of biogenic magnetite, knowledge of magnetosome biosynthesis and biomineralization remains very limited. METHOD: Cells used in this study were cultured in a 7.5-L bioreactor, samples for intermediate capture were taken each certain time interval after the generation of magnetosome biosynthesis condition. High-resolution transmission electron microscopy were used to analyze the detailed structure of magnetosomes. The parameters of the crystal structures were obtained by Fast Fourier Transform analyses. RESULTS: In this study, we identified a novel intermediate phase, ε-Fe2O3, during the magnetite maturation process in MTB via kinetic analysis. Unlike α-Fe2O3, which has been reported as a precursor during magnetosome biosynthesis in MTB before, ε-Fe2O3, due to its thermal instability, is a rare phase with scarce natural abundance. This finding confirmed that ε-Fe2O3 is an important novel intermediate during the biomineralization of magnetosome in MTB, and shed new light on the magnetosome biosynthesis pathway.

A new technology for reducing anastomotic fistula in the neck after esophageal cancer surgery.

BACKGROUND: To explore the risk factors and prevention methods of cervical mechanical anastomotic fistula and stenosis after the radical resection of esophageal cancer. METHODS: From March 2018 to November 2018, 128 patients undergoing mechanical anastomosis of esophageal cancer were selected from the Department of Thoracic Surgery of The First Affiliated Hospital of Zhengzhou University. All the enrolled patients were operated on using the Mckeown method, and a retrospective study was conducted. Data for preoperative and postoperative test indices, intraoperative embedding materials, postoperative complications, and preoperative and postoperative treatment were collected, and the relationship between various factors and the incidence of cervical anastomotic fistula and stenosis was analysed. Univariate analysis was conducted using t tests or Fisher's exact probability method, and multivariate analysis was conducted using logistic regression models. RESULTS: All 128 patients successfully underwent surgery without dying. The enrolled patients were evaluated using the Stooler classification, with 28 patients having grade 0, 41 patients having grade 1, 34 patients having grade 2, 21 patients having grade 3, and 4 patients having grade 4 stenosis. Patients with stenosis of grade 3 or above had obvious choking sensation, which could only be relieved by balloon dilation. Symptoms in all patients with stenosis were relieved by balloon dilation. There were no significant differences between the two groups regarding embedding materials, preoperative choking history, history of alcohol consumption, history of hypertension, history of coronary heart disease, history of diabetes, postoperative calcium concentration, average albumin concentration, average platelet concentration, body mass index, anastomotic fistula, preoperative chemotherapy, postoperative chemotherapy, or postoperative cough (P>0.05). There were significant differences in postoperative reflux (χ2=11.338, P<0.05) and scar constitution (χ2=12.497, P<0.05). The effects of embedding materials in patients with anastomotic fistula were significantly different (χ2=4.372, P<0.05). CONCLUSIONS: Postoperative reflux and scar constitution may be risk factors for postoperative anastomotic stenosis after resection of esophageal cancer. There was almost no difference in the effects on esophageal anastomotic stenosis between embedding materials and the omentum majus, but Neoveil® may have certain advantages in preventing cervical anastomotic fistula, and thus may have certain clinical application value.

AveI, an AtrA homolog of Streptomyces avermitilis, controls avermectin and oligomycin production, melanogenesis, and morphological differentiation.

Streptomyces avermitilis is well known as the producer of anthelmintic agent avermectins, which are widely used in agriculture, veterinary medicine, and human medicine. aveI encodes a TetR-family regulator, which is the homolog of AtrA. It was reported that deletion of aveI caused enhanced avermectin production. In this study, we investigated the regulatory function of the AveI in S. avermitilis. By binding to the 15-nt palindromic sequence in the promoter regions, AveI directly regulates at least 35 genes. AveI represses avermectin production by directly regulating the transcription of the cluster-situated regulator gene aveR and structural genes aveA1, aveA3, and aveD. AveI represses oligomycin production by repressing the CSR gene olmRII and structural genes olmC. AveI activates melanin biosynthesis by activating the expression of melC1C2 operon. AveI activates morphological differentiation by activating the expression of ssgR and ssgD genes, repressing the expression of wblI gene. Besides, AveI regulates many genes involved in primary metabolism, including substrates transport, the metabolism of amino acids, lipids, and carbohydrates. Therefore, AveI functions as a global regulator in S. avermitilis, controls not only secondary metabolism and morphological differentiation, but also primary metabolism.

Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status.

Magnetotactic bacteria (MTB) are a large, polyphyletic group of aquatic microorganisms capable of absorbing large amounts of iron and synthesizing intercellular nano-scaled nanoparticles termed magnetosomes. In our previous transcriptomic studies, we discovered that a novel gene (MGMSRv2_2046, termed as mg2046) in Magnetospirillum gryphiswaldense strain MSR-1 was significantly up-regulated during the period of magnetosome synthesis. In the present study, we constructed a MSR-1 mutant strain with deletion of mg2046 (termed Δmg2046) in order to evaluate the role of this gene in cell physiological status and magnetosome formation process. In comparison with wild-type MSR-1, Δmg2046 showed similar cell growth, but much lower cell magnetic response, smaller number and size of magnetosomes, and reduced iron absorption ability. mg2046 deletion evidently disrupted iron uptake, and redox equilibrium, and strongly inhibited transcription of dissimilatory denitrification pathway genes. Our experimental findings, taken together with results of gene homology analysis, indicate that Mg2046 acts as a positive regulator in MSR-1 under microaerobic conditions, responding to hypoxia signals and participating in regulation of oxygen metabolism, in part as a co-regulator of dissimilatory denitrification pathway with oxygen sensor MgFnr (MGMSRv2_2946, termed as Mg2946). Mg2046 is clearly involved in coupled regulation of cellular oxygen, iron and nitrogen metabolism under micro-aerobic or anaerobic conditions. Our findings help explain how MSR-1 cells initiate dissimilatory denitrification pathway and overcome energy deficiency under microaerobic conditions, and have broader implications regarding bacterial survival and energy metabolism strategies under hypoxia.

Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes.

We demonstrated previously that expression of Rhizomucor miehei lipase (RML) in Pichia pastoris could be significantly increased by addition of gene propeptide, optimized signal peptide codons and manipulation of gene dosage. In this study, effects of various strategies on the protein synthesis and secretion pathways were analyzed. Using nine strains previously constructed, we evaluated cell culture properties, enzymatic activities, and analyzed transcriptional levels of nine genes involved in protein synthesis and secretion pathways by qPCR. We observed that (i) Addition of propeptide decreased lipase folding stress by down-regulated four UPR-related genes. (ii) Signal peptide codons optimization had no effect on host with no change in the nine detected genes. (iii) Folding stress and limited transport capacity produced when rml gene dosage exceed 2. Different limiting factors on lipase expression in strains with different construction strategies were identified. This study provides a theoretical basis for further improving RML by transforming host.

Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae.

Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N2-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (∼38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at δ and HO sites in Saccharomyces cerevisiae chromosome. Of the 15 genes, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nifS, nifU, nifF, nifJ) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.

α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al.

Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-"FeMoco". Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.

Work Patterns of MamXY Proteins during Magnetosome Formation in Magnetospirillum gryphiswaldense MSR-1.

The bacterium Magnetospirillum gryphiswaldense MSR-1 forms nanosized membrane-enclosed organelles termed magnetosomes. The mamXY operon, part of the magnetosome island (MAI), includes the mamY, mamX, mamZ, and ftsZ-like genes, which initiate gene transcription via the same promoter. We used a combination of molecular biological techniques (targeting of cross-linking reagents) and high-resolution mass spectrometry to investigate the coordinated activity of the four MamXY proteins in magnetite biomineralization. The FtsZ-like protein was shown by confocal laser scanning microscopy to be dispersed in the cytoplasm in the early stage of cell growth and then gradually polymerized along the magnetosome chain. Interactions of various pairs of MamXY proteins were observed using a bacterial two-hybrid system. We constructed a recombinant FtsZ-like-overexpressing strain, examined its growth patterns, and extracted magnetosome membrane proteins using a modified SDS/boiling method with BS2G-d0/d4 reagent, which helped stabilize interactions among MamXY proteins. In liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, MamY expression was detected first and remained highest among the four proteins throughout all stages of cell growth. MamX and MamZ expression was detected subsequently. The four proteins displayed coordinated expression patterns during the magnetosome maturation process. Unique peptides discovered in the MamXY protein sequences appeared to constitute "hidden" interaction sites involved in the formation of MamXY complex that helped control magnetosome shape and size.IMPORTANCEmamXY operon genes play an essential role in magnetite biomineralization, participate in redox reactions, and control magnetosome shape and size. However, mechanisms whereby the four MamXY proteins function together in iron oxidoreduction and transport are poorly understood. We used a combination of targeted cross-linking techniques and high-resolution mass spectrometry to elucidate the coordinated activity patterns of the MamXY proteins during magnetite biomineralization. Our findings indicate that the FtsZ-like protein undergoes polymerization and then recruits MamY, MamX, and MamZ in turn, and that these interactions depend on unique peptides present in the protein sequences. A hypothetical model of the functionalities of these proteins is proposed that accounts for the findings and provides a basis for further studies of coordination among magnetosome island (MAI) gene clusters during the process of magnetosome formation.

IdeR, a DtxR Family Iron Response Regulator, Controls Iron Homeostasis, Morphological Differentiation, Secondary Metabolism, and the Oxidative Stress Response in Streptomyces avermitilis.

Iron, an essential element for microorganisms, functions as a vital cofactor in a wide variety of key metabolic processes. On the other hand, excess iron may have toxic effects on bacteria by catalyzing the formation of reactive oxygen species through the Fenton reaction. The prevention of iron toxicity requires the precise control of intracellular iron levels in bacteria. Mechanisms of iron homeostasis in the genus Streptomyces (the producers of various antibiotics) are poorly understood. Streptomyces avermitilis is the industrial producer of avermectins, which are potent anthelmintic agents widely used in medicine, agriculture, and animal husbandry. We investigated the regulatory role of IdeR, a DtxR family regulator, in S. avermitilis In the presence of iron, IdeR binds to a specific palindromic consensus sequence in promoters and regulates 14 targets involved in iron metabolism (e.g., iron acquisition, iron storage, heme metabolism, and Fe-S assembly). IdeR also directly regulates 12 targets involved in other biological processes, including morphological differentiation, secondary metabolism, carbohydrate metabolism, and the tricarboxylic acid (TCA) cycle. ideR transcription is positively regulated by the peroxide-sensing transcriptional regulator OxyR. A newly constructed ideR deletion mutant (DideR) was found to be less responsive to iron levels and more sensitive to H2O2 treatment than the wild-type strain, indicating that ideR is essential for oxidative stress responses. Our findings, taken together, demonstrate that IdeR plays a pleiotropic role in the overall coordination of metabolism in Streptomyces spp. in response to iron levels.IMPORTANCE Iron is essential to almost all organisms, but in the presence of oxygen, iron is both poorly available and potentially toxic. Streptomyces species are predominantly present in soil where the environment is complex and fluctuating. So far, the mechanism of iron homeostasis in Streptomyces spp. remains to be elucidated. Here, we characterized the regulatory role of IdeR in the avermectin-producing organism S. avermitilis IdeR maintains intracellular iron levels by regulating genes involved in iron absorption and storage. IdeR also directly regulates morphological differentiation, secondary metabolism, and central metabolism. ideR is under the positive control of OxyR and is indispensable for an efficient response to oxidative stress. This investigation uncovered that IdeR acts as a global regulator coordinating iron homeostasis, morphological differentiation, secondary metabolism, and oxidative stress response in Streptomyces species. Elucidation of the pleiotropic regulation function of IdeR provides new insights into the mechanisms of how Streptomyces spp. adapt to the complex environment.