Publications by authors named "Dongru Qiu"

27 Publications

  • Page 1 of 1

Changes of microbial community structure during the initial stage of biological clogging in horizontal subsurface flow constructed wetlands.

Bioresour Technol 2021 Oct 12;337:125405. Epub 2021 Jun 12.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.

The clogging is a universal problem in constructed wetlands, where microorganisms play an essential role. However, the implication of micro-organism variation due to the clogging is not clear. Four horizontal subsurface flow constructed wetlands (HFCWs) were designed and operated to simulate the process of clogging. The wetland treatment performance and microbial community variation were investigated by regularly monitoring. Results showed the substrate filtration rate and the total phosphorous (TP) removal efficiency consistently decreased and the chemical oxygen demand (COD) and total nitrogen (TN) removal efficiency were at the range of 50%-85% and 10-20%, respectively. The sequencing results indicated that the clogging could affect the richness of bacterial community. The bacterial variation could be attributed to the dissolved oxygen decreasing and organic matter accumulation in the initial clogging period. These findings are expected to provide some theoretical reference for developing the biological methods to indicate the initial clogging in constructed wetlands.
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http://dx.doi.org/10.1016/j.biortech.2021.125405DOI Listing
October 2021

Dissimilatory Nitrate Reduction to Ammonium (DNRA) and Denitrification Pathways Are Leveraged by Cyclic AMP Receptor Protein (CRP) Paralogues Based on Electron Donor/Acceptor Limitation in Shewanella loihica PV-4.

Appl Environ Microbiol 2021 01 4;87(2). Epub 2021 Jan 4.

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

Under anoxic conditions, many bacteria, including strain PV-4, could use nitrate as an electron acceptor for dissimilatory nitrate reduction to ammonium (DNRA) and/or denitrification. Previous and current studies have shown that DNRA is favored under higher ambient carbon-to-nitrogen (C/N) ratios, whereas denitrification is upregulated under lower C/N ratios, which is consistent with our bioenergetics calculations. Interestingly, computational analyses indicate that the common cyclic AMP receptor protein (designated CRP1) and its paralogue CRP2 might both be involved in the regulation of two competing dissimilatory nitrate reduction pathways, DNRA and denitrification, in PV-4 and several other denitrifying species. To explore the regulatory mechanism underlying the dissimilatory nitrate reduction (DNR) pathways, nitrate reduction of a series of in-frame deletion mutants was analyzed under different C/N ratios. Deletion of could accelerate the reduction of nitrite to NO under both low and high C/N ratios. CRP1 is not required for denitrification and actually suppresses production of NO and NO gases. Deletion of either of the NO-forming nitrite reductase genes or blocked production of NO gas. Furthermore, real-time PCR and electrophoretic mobility shift assays (EMSAs) demonstrated that the transcription levels of DNRA-relevant genes such as -β (), , and were upregulated by CRP1, while transcription was dependent on CRP2. There are tradeoffs between the different physiological roles of nitrate/lactate, as nitrogen nutrient/carbon source and electron acceptor/donor and CRPs may leverage dissimilatory nitrate reduction pathways for maximizing energy yield and bacterial survival under ambient environmental conditions. Some microbes utilize different dissimilatory nitrate reduction (DNR) pathways, including DNR to ammonia (DNRA) and denitrification pathways, for anaerobic respiration in response to ambient carbon/nitrogen ratio changes. Large-scale industrial nitrogen fixation and fertilizer application raise the concern of emission of NO, a stable gas with potent global warming potential, as consequence of microbial respiration, thereby aggravating global warming and climate change. However, little is known about the molecular mechanism underlying the choice of two competing DNR pathways. We demonstrate that the global regulator CRP1, which is widely encoded in bacteria, is required for DNRA in PV-4 strain, while the CRP2 paralogue is required for transcription of the nitrite reductase gene for denitrification. Sufficient carbon source lead to the predominance of DNRA, while carbon source/electron donor deficiency may result in an incomplete denitrification process, raising the concern of high levels of NO emission from nitrate-rich and carbon source-poor waters and soils.
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http://dx.doi.org/10.1128/AEM.01964-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7783327PMC
January 2021

sp. nov., a novel member of isolated from activated sludge collected in Hong Kong.

Int J Syst Evol Microbiol 2020 Nov;70(11):5799-5805

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China.

A floc-forming bacterial strain, designated HKLI-1, was isolated from the activated sludge of a municipal sewage treatment plant in Hong Kong SAR, PR China. Cells of this strain were Gram-stain-negative, strictly aerobic, catalase- and oxidase-positive, rod-shaped and motile by means of a single polar flagellum. Growth occurred at 18-37 °C (optimum, 28 °C), pH 5.5-9.0 (optimum, pH 7.5) and with 0-8.0 % (w/v) NaCl (optimum, 1-1.5 %) concentration. The major fatty acids of strain HKLI-1 were C and summed feature 3 (C ω7 and/or C ω6). The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and three unidentified lipids. The DNA G+C content was 63.5 mol% from whole genomic sequence analysis. Based on the results of 16S rRNA gene sequences analysis, this strain should be assigned to the genus and is closely related to DQS-4 (94.93 % 16S rRNA gene sequence pairwise similarity), MF63 (94.91 %) and SWub3 (94.01 %), but separate from them by large distances in different phylogenetic trees. Based on whole genome analysis, the orthologous average nucleotide identity and DNA-DNA hybridization values against four of the closest relatives were 73.03-74.83 and 17.2-23.0 %, respectively. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrated that strain HKLI-1 could be distinguished from its phylogenetically related species, and that this strain represented a novel species within the genus , for which the name sp. nov. is proposed. The type strain is HKLI-1 (= 72659=CCTCC AB 2019312).
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http://dx.doi.org/10.1099/ijsem.0.004476DOI Listing
November 2020

Gene expression abundance dictated exopolysaccharide modification in Rhizobium radiobacter SZ4S7S14 as the cell's response to salt stress.

Int J Biol Macromol 2020 Dec 12;164:4339-4347. Epub 2020 Sep 12.

Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, Xinjiang, PR China; State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Xinggang West Road, Guangzhou 510275, PR China.

A molecular and metabolic behaviour of EPS-producing and salt-tolerant bacterium Rhizobium radiobacter SZ4S7S14 along with its practical application in salt-stress was investigated. The research target was identification and expression profiles of a large EPS biosynthesis gene cluster, possible structural modification of EPS under salt-stress effect and analysis of the gene(s) relative expression and structural modification correlation. As expected, transposons insertions were identified within or near the coding regions of exoK and exoM, previously known large gene cluster that is required for EPS I synthesis. Different expression levels of exoK and exoM in different salt-stress models resulted in structural modification of EPS, which was seen basically in monomers molar ratio. As a result of downregulation of the genes the strain produced EPS samples with monomers ratio: (1) Glu:Man:Gal:Xyl:Ara:Rha:Rib = 31.21:3.02:2.77:1:0.91:0.64:0.41 (in 0.25% NaCl); (2) Glu:Man:Gal:Xyl:Ara:Rha:Rib = 7.65:1:0.69:0.22:0.2:0.16:0.1 (in 0.5% NaCl); (3) Glu:Man:Gal:Ara:Xyl:Rha:Rib = 9.39:1.89:1:0.58:0.52:0.46:0.26 (in 1% NaCl); and (4) Glu:Man:Ara:Xyl:Rib:Gal = 7.9:2:2:1.58:1.1:1 (in 2.0% NaCl), whereas in control (without NaCl): Glc:Man:Gal:Xyl:Ara:Rha:Rib = 11.66:1:0.90:0.37:0.37:0.15:0.14. It was found that, salt-stress not only leads to downregulation of a large EPS biosynthesis gene cluster, including exoK and exoM genes, but also impacting on their relative expression degree, re-groups of the monomers within the EPS matrix and dictates molar ratio of the monosaccharides in the final metabolite.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.09.038DOI Listing
December 2020

Production of Highly Active Extracellular Amylase and Cellulase From ZIM3 and a Recombinant Strain With a Potential Application in Tobacco Fermentation.

Front Microbiol 2020 21;11:1539. Epub 2020 Jul 21.

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.

In this study, a series of bacteria capable of degrading starch and cellulose were isolated from the aging flue-cured tobacco leaves. Remarkably, there was a thermophilic bacterium, ZIM3, that can simultaneously degrade both starch and cellulose at a wide range of temperature and pH values. Genome sequencing, comparative genomics analyses, and enzymatic activity assays showed that the ZIM3 strain expressed a variety of highly active plant biomass-degrading enzymes, such as the amylase AmyE1 and cellulase CelE1. The and PhoA-fusion assays indicated that these enzymes degrading complex plant biomass into fermentable sugars were secreted into ambient environment to function. Besides, the amylase and cellulase activities were further increased by three- to five-folds by using overexpression. Furthermore, a fermentation strategy was developed and the biodegradation efficiency of the starch and cellulose in the tobacco leaves were improved by 30-48%. These results reveal that ZIM3 and the recombinant strain exhibited high amylase and cellulase activities for efficient biodegradation of starch and cellulose in tobacco and could potentially be applied for industrial tobacco fermentation.
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http://dx.doi.org/10.3389/fmicb.2020.01539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385192PMC
July 2020

Disodium 2-oxoglutarate promotes carbon flux into astaxanthin and fatty acid biosynthesis pathways in Haematococcus.

Bioresour Technol 2020 Mar 13;299:122612. Epub 2019 Dec 13.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China. Electronic address:

Improving carbon availability in astaxanthin production is pivotal in Haematococcus industry. In this study, disodium 2-oxoglutarate (2-OG-2Na) was observed to be a potential carbon regulator to increase the astaxanthin content. To illustrate its efficacy in astaxanthin production, key genes and enzyme were analyzed. Upon 2-OG-2Na treatment, genes ipi, bkt and crtR-b were up regulated, concomitantly, carotenoids and astaxanthin content increased by 15.4% and 14.0% at 120 h, respectively; additionally, Acetyl-CoA carboxylase was activated, consistent with 1.27-fold increase in fatty acids content. PUFAs increased earlier as fatty acids assembly gene fad was up-regulated to 20.56. It was also found that cell division was not compromised. Altogether, it was suggested that increased carbon skeletons were re-directed into the astaxanthin and fatty acids biosynthesis pathway. Furthermore, 2-OG-2Na was applied in ten Haematococcus strains. Of these strains, astaxanthin contents were accelerated with average net increase of 10.48%, exhibiting a scalable paradigm for commercial production.
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http://dx.doi.org/10.1016/j.biortech.2019.122612DOI Listing
March 2020

Effects of maifanite on growth, physiological and phytochemical process of submerged macrophytes Vallisneria spiralis.

Ecotoxicol Environ Saf 2020 Feb 21;189:109941. Epub 2019 Nov 21.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; School of Resources and Environmental Engineering, Wuhan University of Technology, Wuhan, 430070, China.

The restoration of submerged plants is critical for the reconstruction of eutrophic lake ecosystems. The growth of submerged plants is influenced by many factors. For the first time in this study, the effects of silicate-mineral maifanite supplement on the growth, physiological and phytochemical process of Vallisneria spiralis (V. spiralis) were investigated by an outdoor PVC barrel experiment, to provide a technical reference for further applications in aquatic ecological restoration. The results show that the maifanite could significantly promote the growth of V. spiralis. Specifically, the biomass, height, number of leaves, leaf width, root length, and root activity of V. spiralis in the maifanite-supplemented group were better than those of the control (P < 0.05). Moreover, the modified maifanite group performed better than the raw maifanite group (P < 0.05). The photosynthetic pigment, root activity, and the malondialdehyde and peroxidase activity of the maifanite-treated V. spiralis were better than those of the control to some extent. It was found that maifanite contained abundant major and trace elements, which are required for the growth of V. spiralis. It is concluded that maifanite is beneficial to the growth of V. spiralis and can be further applied to the ecological restoration of eutrophic lakes.
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http://dx.doi.org/10.1016/j.ecoenv.2019.109941DOI Listing
February 2020

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins.

BMC Microbiol 2019 07 30;19(1):173. Epub 2019 Jul 30.

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, Hubei Province, China.

Background: Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1.

Result: In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome.

Conclusion: Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.
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http://dx.doi.org/10.1186/s12866-019-1549-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664582PMC
July 2019

Both widespread PEP-CTERM proteins and exopolysaccharides are required for floc formation of Zoogloea resiniphila and other activated sludge bacteria.

Environ Microbiol 2018 05 26;20(5):1677-1692. Epub 2018 Mar 26.

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.

Bacterial floc formation plays a central role in the activated sludge (AS) process, which has been widely utilized for sewage and wastewater treatment. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. This study demonstrates an additional requirement for a PEP-CTERM protein in Zoogloea resiniphila, a dominant AS bacterium harboring a large exopolysaccharide biosynthesis gene cluster. Two members of a wide-spread family of high copy number-per-genome PEP-CTERM genes, transcriptionally regulated by the RpoN sigma factor and accessory PrsK-PrsR two-component system and at least one of these, pepA, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling. Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated and secreted exopolysaccharides were released into the growth broth in soluble form. Overexpression of PepA could circumvent the requirement of rpoN, prsK and prsR for the floc-forming phenotype by fixing the exopolysaccharides to bacterial cells. However, overexpression of PepA, which underwent post-translational modifications, could not rescue the long-rod morphology of the rpoN mutant. Consistently, PEP-CTERM genes and exopolysaccharide biosynthesis gene cluster are present in the genome of the floc-forming Nitrospira comammox and Mitsuaria strain as well as many other AS bacteria.
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http://dx.doi.org/10.1111/1462-2920.14080DOI Listing
May 2018

RpoN (σ) Is Required for Floc Formation but Not for Extracellular Polysaccharide Biosynthesis in a Floc-Forming Aquincola tertiaricarbonis Strain.

Appl Environ Microbiol 2017 07 30;83(14). Epub 2017 Jun 30.

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

Some bacteria are capable of forming flocs, in which bacterial cells become self-flocculated by secreted extracellular polysaccharides and other biopolymers. The floc-forming bacteria play a central role in activated sludge, which has been widely utilized for the treatment of municipal sewage and industrial wastewater. Here, we use a floc-forming bacterium, RN12, as a model to explore the biosynthesis of extracellular polysaccharides and the regulation of floc formation. A large gene cluster for exopolysaccharide biosynthesis and a gene encoding the alternative sigma factor RpoN1, one of the four paralogues, have been identified in floc formation-deficient mutants generated by transposon mutagenesis, and the gene functions have been further confirmed by genetic complementation analyses. Interestingly, the biosynthesis of exopolysaccharides remained in the -disrupted flocculation-defective mutants, but most of the exopolysaccharides were secreted and released rather than bound to the cells. Furthermore, the expression of exopolysaccharide biosynthesis genes seemed not to be regulated by RpoN1. Taken together, our results indicate that RpoN1 may play a role in regulating the expression of a certain gene(s) involved in the self-flocculation of bacterial cells but not in the biosynthesis and secretion of exopolysaccharides required for floc formation. Floc formation confers bacterial resistance to predation of protozoa and plays a central role in the widely used activated sludge process. In this study, we not only identified a large gene cluster for biosynthesis of extracellular polysaccharides but also identified four paralogues, one of which () is required for floc formation in RN12. In addition, this RpoN sigma factor regulates the transcription of genes involved in biofilm formation and swarming motility, as previously shown in other bacteria. However, this RpoN paralogue is not required for the biosynthesis of exopolysaccharides, which are released and dissolved into culture broth by the mutant rather than remaining tightly bound to cells, as observed during the flocculation of the wild-type strain. These results indicate that floc formation is a regulated complex process, and other yet-to-be identified RpoN1-dependent factors are involved in self-flocculation of bacterial cells via exopolysaccharides and/or other biopolymers.
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http://dx.doi.org/10.1128/AEM.00709-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494619PMC
July 2017

Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria.

Water Res 2016 10 1;102:494-504. Epub 2016 Jul 1.

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Electronic address:

Activated sludge (AS) process has been widely utilized for municipal sewage and industrial wastewater treatment. Zoolgoea and its related floc-forming bacteria are required for formation of AS flocs which is the key to gravitational effluent-and-sludge separation and AS recycling. However, little is known about the genetics, biochemistry and physiology of Zoogloea and its related bacteria. This report deals with the comparative genomic analyses on two Zoogloea resiniphila draft genomes and the closely related proteobacterial species commonly found in AS. In particular, the metabolic processes involved in removal of organic matters, nitrogen and phosphorus were analyzed. Furthermore, it is revealed that a large gene cluster, encoding eight glycosyltransferases and other proteins involved in biosynthesis and export of extracellular polysaccharides (EPS), was required for floc formation. One of the two asparagine synthase paralogues, associated with this EPS biosynthesis gene cluster, was required for floc formation in Zoogloea. Similar EPS biosynthesis gene cluster(s) were identified in the genome of other AS proteobacteria including polyphosphate-accumulating Candidatus Accumulibacter phosphatis (CAP) and nitrifying Nitrosopira and Nitrosomonas bacteria, but the gene composition varies interspecifically and intraspecifically. Our results indicate that floc formation of desired AS bacteria, including CAP strains, facilitate their recruitment into AS and gradual enrichment via repeated AS settling and recycling processes.
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http://dx.doi.org/10.1016/j.watres.2016.06.058DOI Listing
October 2016

Differential Regulation of the Two Ferrochelatase Paralogues in Shewanella loihica PV-4 in Response to Environmental Stresses.

Appl Environ Microbiol 2016 09 15;82(17):5077-88. Epub 2016 Aug 15.

Institute for Environmental Genomics, Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, China

Unlabelled: Determining the function and regulation of paralogues is important in understanding microbial functional genomics and environmental adaptation. Heme homeostasis is crucial for the survival of environmental microorganisms. Most Shewanella species encode two paralogues of ferrochelatase, the terminal enzyme in the heme biosynthesis pathway. The function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, were investigated in Shewanella loihica PV-4. The disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), the precursor to heme, and decreased intracellular heme levels. hemH1 was constitutively expressed, and the expression of hemH2 increased when hemH1 was disrupted. The transcription of hemH1 was regulated by the housekeeping sigma factor RpoD and potentially regulated by OxyR, while hemH2 appeared to be regulated by the oxidative stress-associated sigma factor RpoE2. When an oxidative stress condition was mimicked by adding H2O2 to the medium or exposing the culture to light, PPIX accumulation was suppressed in the ΔhemH1 mutant. Consistently, transcriptome analysis indicated enhanced iron uptake and suppressed heme synthesis in the ΔhemH1 mutant. These data indicate that the two paralogues are functional in the heme synthesis pathway but regulated by environmental conditions, providing insights into the understanding of bacterial response to environmental stresses and a great potential to commercially produce porphyrin compounds.

Importance: Shewanella is capable of utilizing a variety of electron acceptors for anaerobic respiration because of the existence of multiple c-type cytochromes in which heme is an essential component. The cytochrome-mediated electron transfer across cellular membranes could potentially be used for biotechnological purposes, such as electricity generation in microbial fuel cells and dye decolorization. However, the mechanism underlying the regulation of biosynthesis of heme and cytochromes is poorly understood. Our study has demonstrated that two ferrochelatase genes involved in heme biosynthesis are differentially regulated in response to environmental stresses, including light and reactive oxygen species. This is an excellent example showing how bacteria have evolved to maintain cellular heme homeostasis. More interestingly, the high yields of extracellular protoporphyrin IX by the Shewanella loihica PV-4 mutants could be utilized for commercial production of this valuable chemical via bacterial fermentation.
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http://dx.doi.org/10.1128/AEM.00203-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988212PMC
September 2016

Truncated type IV pilin PilA(108) activates the intramembrane protease AlgW to cleave MucA and PilA(108) itself in vitro.

Arch Microbiol 2016 Nov 6;198(9):885-92. Epub 2016 Jun 6.

Institute of Hydrobiology, Chinese Academy of Sciences, 7 South Donghu Road, Wuchang District, Wuhan, 430072, China.

For alginate production in Pseudomonas aeruginosa, the intramembrane protease AlgW must be activated to cleave the periplasmic domain of anti-sigma factor MucA for release of the sequestered ECF sigma factor AlgU. Previously, we reported that three tandem point mutations in the pilA gene, resulting in a truncated type IV pilin termed PilA(108) with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF), induced mucoidy in strain PAO579. In this study, we purified PilA(108) protein and synthesized a peptide 'SGAGDITFTF' corresponding to C-terminus of PilA(108) and found they both caused the degradation of MucA by AlgW. Interestingly, AlgW could also cleave PilA(108) between alanine(62) and glycine(63) residues. Overexpression of the recombinant FTF motif-bearing MucE protein, originally a small periplasmic polypeptide with the C-terminal motif WVF, could induce mucoid conversion in the PAO1 strain. In all, our results provided a model of activation of AlgW by another protein ending with proper motifs. Our data suggest that in addition to MucA cleavage, AlgW may cleave other substrates.
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http://dx.doi.org/10.1007/s00203-016-1248-yDOI Listing
November 2016

Functional roles of CymA and NapC in reduction of nitrate and nitrite by Shewanella putrefaciens W3-18-1.

Microbiology (Reading) 2016 06 23;162(6):930-941. Epub 2016 Mar 23.

Institute of hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China.

Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.
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http://dx.doi.org/10.1099/mic.0.000285DOI Listing
June 2016

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis.

BMC Microbiol 2015 Feb 18;15:34. Epub 2015 Feb 18.

Institute of Hydrobiology, Chinese Academy of Sciences, 7 South Donghu Road, Wuchang District, Wuhan, 430072, China.

Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.

Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.

Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.
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http://dx.doi.org/10.1186/s12866-015-0357-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336711PMC
February 2015

Effects of intermediate metabolite carboxylic acids of TCA cycle on Microcystis with overproduction of phycocyanin.

Environ Sci Pollut Res Int 2015 Apr 24;22(7):5531-7. Epub 2014 Oct 24.

Institute of Hydrobiology, The Chinese Academy of Sciences and University of Chinese Academy of Sciences, Wuhan, 430072, China.

Toxic Microcystis species are the main bloom-forming cyanobacteria in freshwaters. It is imperative to develop efficient techniques to control these notorious harmful algal blooms (HABs). Here, we present a simple, efficient, and environmentally safe algicidal way to control Microcystis blooms, by using intermediate carboxylic acids from the tricarboxylic acid (TCA) cycle. The citric acid, alpha-ketoglutaric acid, succinic acid, fumaric acid, and malic acid all exhibited strong algicidal effects, and particularly succinic acid could cause the rapid lysis of Microcystis in a few hours. It is revealed that the Microcystis-lysing activity of succinic acid and other carboxylic acids was due to their strong acidic activity. Interestingly, the acid-lysed Microcystis cells released large amounts of phycocyanin, about 27-fold higher than those of the control. On the other hand, the transcription of mcyA and mcyD of the microcystin biosynthesis operon was not upregulated by addition of alpha-ketoglutaric acid and other carboxylic acids. Consider the environmental safety of intermediate carboxylic acids. We propose that administration of TCA cycle organic acids may not only provide an algicidal method with high efficiency and environmental safety but also serve as an applicable way to produce and extract phycocyanin from cyanobacterial biomass.
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http://dx.doi.org/10.1007/s11356-014-3730-xDOI Listing
April 2015

Roles of UndA and MtrC of Shewanella putrefaciens W3-18-1 in iron reduction.

BMC Microbiol 2013 Nov 25;13:267. Epub 2013 Nov 25.

State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, China.

Background: The completion of genome sequencing in a number of Shewanella species, which are most renowned for their metal reduction capacity, offers a basis for comparative studies. Previous work in Shewanella oneidensis MR-1 has indicated that some genes within a cluster (mtrBAC-omcA-mtrFED) were involved in iron reduction. To explore new features of iron reduction pathways, we experimentally analyzed Shewanella putrefaciens W3-18-1 since its gene cluster is considerably different from that of MR-1 in that the gene cluster encodes only four ORFs.

Results: Among the gene cluster, two genes (mtrC and undA) were shown to encode c-type cytochromes. The ΔmtrC deletion mutant revealed significant deficiencies in reducing metals of Fe2O3, α-FeO(OH), β-FeO(OH), ferric citrate, Mn(IV) and Co(III), but not organic compounds. In contrast, no deficiency of metal reduction was observed in the ΔundA deletion mutant. Nonetheless, undA deletion resulted in progressively slower iron reduction in the absence of mtrC and fitness loss under the iron-using condition, which was indicative of a functional role of UndA in iron reduction.

Conclusions: These results provide physiological and biochemical evidences that UndA and MtrC of Shewanella putrefaciens W3-18-1 are involved in iron reduction.
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http://dx.doi.org/10.1186/1471-2180-13-267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222724PMC
November 2013

Combined genomics and experimental analyses of respiratory characteristics of Shewanella putrefaciens W3-18-1.

Appl Environ Microbiol 2013 Sep 28;79(17):5250-7. Epub 2013 Jun 28.

Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.

It has previously been shown that the Shewanella putrefaciens W3-18-1 strain produces remarkably high current in microbial fuel cells (MFCs) and can form magnetite at 0°C. To explore the underlying mechanisms, we developed a genetic manipulation method by deleting the restriction-modification system genes of the SGI1 (Salmonella genome island 1)-like prophage and analyzed the key genes involved in bacterial respiration. W3-18-1 has less respiratory flexibility than the well-characterized S. oneidensis MR-1 strain, as it possesses fewer cytochrome c genes and lacks the ability to oxidize sulfite or reduce dimethyl sulfoxide (DMSO) and timethylamine oxide (TMAO). W3-18-1 lacks the hydrogen-producing Fe-only hydrogenase, and the hydrogen-oxidizing Ni-Fe hydrogenase genes were split into two separate clusters. Two periplasmic nitrate reductases (NapDAGHB and NapDABC) were functionally redundant in anaerobic growth of W3-18-1 with nitrate as the electron acceptor, though napDABC was not regulated by Crp. Moreover, nitrate respiration started earlier in W3-18-1 than in MR-1 (with NapDAGHB only) under microoxic conditions. These results indicate that Shewanella putrefaciens W3-18-1 is well adapted to habitats with higher oxygen levels. Taken together, the results of this study provide valuable insights into bacterial genome evolution.
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http://dx.doi.org/10.1128/AEM.00619-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753967PMC
September 2013

[Essential genes, minimal genome and synthetic cell of bacteria: a review].

Authors:
Dongru Qiu

Sheng Wu Gong Cheng Xue Bao 2012 May;28(5):540-9

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei, China.

Single-cell prokaryotes represent a simple and primitive cellular life form. The identification of the essential genes of bacteria and the minimal genome for the free-living cellular life could provide insights into the origin, evolution, and essence of life forms. The principles, methodology, and recent progresses in the identification of essential genes and minimal genome and the creation of synthetic cells are reviewed and particularly the strategies for creating the minimal genome and the potential applications are introduced.
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May 2012

The Pseudomonas aeruginosa sensor kinase KinB negatively controls alginate production through AlgW-dependent MucA proteolysis.

J Bacteriol 2009 Apr 23;191(7):2285-95. Epub 2009 Jan 23.

Department of Biochemistry, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia 25755-9320, USA.

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-sigma factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P(algU) and P(algD). Deletion of alternative sigma factor RpoN (sigma(54)) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.
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http://dx.doi.org/10.1128/JB.01490-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655532PMC
April 2009

PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria.

Appl Environ Microbiol 2008 Dec 10;74(23):7422-6. Epub 2008 Oct 10.

Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia 25755-9320, USA.

We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P(BAD) promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.
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http://dx.doi.org/10.1128/AEM.01369-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592904PMC
December 2008

ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa.

Microbiology (Reading) 2008 Jul;154(Pt 7):2119-2130

Department of Pediatrics, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV 25701-3655, USA.

Overproduction of the exopolysaccharide alginate and conversion to a mucoid phenotype in Pseudomonas aeruginosa are markers for the onset of chronic lung infection in cystic fibrosis (CF). Alginate production is regulated by the extracytoplasmic function (ECF) sigma factor AlgU/T and the cognate anti-sigma factor MucA. Many clinical mucoid isolates carry loss-of-function mutations in mucA. These mutations, including the most common mucA22 allele, cause C-terminal truncations in MucA, indicating that an inability to regulate AlgU activity by MucA is associated with conversion to the mucoid phenotype. Here we report that a mutation in a stable mucoid strain derived from the parental strain PAO1, designated PAO581, that does not contain the mucA22 allele, was due to a single-base deletion in mucA (DeltaT180), generating another type of C-terminal truncation. A global mariner transposon screen in PAO581 for non-mucoid isolates led to the identification of three regulators of alginate production, clpP (PA1801), clpX (PA1802), and a clpP paralogue (PA3326, designated clpP2). The PAO581 null mutants of clpP, clpX and clpP2 showed decreased AlgU transcriptional activity and an accumulation of haemagglutinin (HA)-tagged N-terminal MucA protein with an apparent molecular mass of 15 kDa. The clpP and clpX mutants of a CF mucoid isolate revert to the non-mucoid phenotype. The ClpXP and ClpP2 proteins appear to be part of a proteolytic network that degrades the cytoplasmic portion of truncated MucA proteins to release the sequestered AlgU, which drives alginate biosynthesis.
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http://dx.doi.org/10.1099/mic.0.2008/017368-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995304PMC
July 2008

Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa.

Proc Natl Acad Sci U S A 2007 May 30;104(19):8107-12. Epub 2007 Apr 30.

Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755-9320, USA.

Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF sigma factor AlgU and its cognate anti-sigma factor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli sigma(E) except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.
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http://dx.doi.org/10.1073/pnas.0702660104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876579PMC
May 2007

Sustained MAPK activation is dependent on continual NGF receptor regeneration.

Dev Growth Differ 2004 Oct;46(5):393-403

Institute for Advanced Biosciences, Keio University, 14-1, Baba, Tsuruoka, Yamagata, 997-0035, Japan.

It still remains intriguing how signal specificity is achieved when different signals are relayed by the common intracellular signal transduction pathways. A well documented example for signal specificity determination is found in rat phaeochromocytoma PC12 cells where epidermal growth factor (EGF) stimulation produces a transient mitogen-activated protein kinase (MAPK) activation and leads to cell proliferation while nerve growth factor (NGF) initiates a sustained MAPK activation and induces cell differentiation. In this simulation, we demonstrated that NGF-induced sustained MAPK activation may mainly depend on continual regeneration of NGF receptors and that the presence of a small pool of surface receptors is enough to maintain a sustained MAPK activation. On the other hand, MAPK activation is not significantly sensitive to the half-life of internalized receptors and the levels of NGF-specific MAPK phosphatase MAP kinase phosphatase-3 (MKP-3), though cytoplasmic persistence of internalized NGF-bound receptors and the MKP-3 dependent feedback control also contribute to the sustaining of MAPK activation. These results are consistent with the recent experimental evidence that persistent tyrosine receptor kinase A (TrkA) activity is necessary to maintain transcription in the differentiating PC12 cells (Chang et al. 2003) and a sustained Src kinase activity is detected in response to NGF stimulation (Gatti 2003). It is suggested that sustained or transient MAPK activation induced by different growth factor and neurotrophins, which is crucial to their signaling specificity, could be satisfactorily accounted for by their specific receptor turnover kinetics rather than by the activation of specific downstream signaling cascades.
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http://dx.doi.org/10.1111/j.1440-169x.2004.00756.xDOI Listing
October 2004

Comparative analysis of physical maps of four Bacillus subtilis (natto) genomes.

Appl Environ Microbiol 2004 Oct;70(10):6247-56

Institute for Advanced Biosciences and Bioinformatics Program, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.

The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure. Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed. No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B. subtilis strain Marburg 168. However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SP beta, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types. The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.
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http://dx.doi.org/10.1128/AEM.70.10.6247-6256.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC522138PMC
October 2004

[Effects of rehabilitation of submerged macrophytes on nutrient level of a eutrophic lake].

Ying Yong Sheng Tai Xue Bao 2003 Aug;14(8):1351-3

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.

The effects of rehabilitation of submerged aquatic vegetation on nutrient level of eutrophic waters were investigated by using large-scale enclosure system installed in a hyper-eutrophicated lake, Lake Donghu of Wuhan. The concentrations of total nitrogen (TN) and total phosphorus (TP) of the enclosures with artificially recovered submerged macrophytes were significantly lower than those of the macrophyte-free control enclosure and the lake water, and the TP of macrophyte enclosure was found to be around 0.1 mg.L-1 and relatively stable. The concentration of ammonia and nitrite of the macrophyte enclosures were lower than those of the control and the lake water. However, the nitrate concentrations did not differ among the various water bodies. It was demonstrated that re-establishment of submerged macrophytes could efficiently reduce the nutrient level and thus control the overgrowth of the undesirable algae and improve water quality in the eutrophic lakes.
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August 2003

Construction of physical maps of Bacillus subtilis (natto) strains.

Nucleic Acids Res Suppl 2003 (3):207-8

Institute for Advanced Biosciences, Bioinformatics program, Graduate School of Governance and Media, Keio University, Tsuruoka, Yamagata 997-0017, Japan.

The putative physical map of a Bacillus subtilis (natto) strain has been constructed to demonstrate the difference from the strain B. subtilis Marburg. B. subtilis (natto) strains are closely related to the Marburg 168trpC2 strain whose genome was sequenced. Their genome size and organization are also assumed to be similar. The putative SfiI and I-CeuI physical map of a natto strain BEST195 revealed that structure of most genome region is similar to that of 168 but some highly variable chromosome regions are also identified.
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http://dx.doi.org/10.1093/nass/3.1.207DOI Listing
October 2003
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