Arsenic effectively improves the degradation of fluorene by Rhodococcus sp. 2021 under the combined pollution of arsenic and fluorene.

The performance of bacterial strains in executing degradative functions under the coexistence of heavy metals/heavy metal-like elements and organic contaminants is understudied. In this study, we isolated a fluorene-degrading bacterium, highly arsenic-resistant, designated as strain 2021, from contaminated soil at the abandoned site of an old coking plant. It was identified as a member of the genus Rhodococcus sp. strain 2021 exhibited efficient fluorene-degrading ability under optimal conditions of 400 mg/L fluorene, 30 °C, pH 7.0, and 250 mg/L trivalent arsenic. It was noted that the addition of arsenic could promote the growth of strain 2021 and improve the degradation of fluorene - a phenomenon that has not been described yet. The results further indicated that strain 2021 can oxidize As3+ to As5+; here, approximately 13.1% of As3+ was converted to As5+ after aerobic cultivation for 8 days at 30 °C. The addition of arsenic could greatly up-regulate the expression of arsR/A/B/C/D and pcaG/H gene clusters involved in arsenic resistance and aromatic hydrocarbon degradation; it also aided in maintaining the continuously high expression of cstA that codes for carbon starvation protein and prmA/B that codes for monooxygenase. These results suggest that strain 2021 holds great potential for the bioremediation of environments contaminated by a combination of arsenic and polycyclic aromatic hydrocarbons. This study provides new insights into the interactions among microbes, as well as inorganic and organic pollutants.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Alcaligenes ammonioxydans HO-1 antagonizes Bacillus velezensis via hydroxylamine-triggered population response.

Antagonism is a common behavior seen between microbes in nature. Alcaligenes ammonioxydans HO-1 converts ammonia to nitrogen under aerobic conditions, which leads to the accumulation of extracellular hydroxylamine (HA), providing pronounced growth advantages against many bacterial genera, including Bacillus velezensis V4. In contrast, a mutant variant of A. ammonioxydans, strain 2-29, that cannot produce HA fails to antagonize other bacteria. In this article, we demonstrate that cell-free supernatants derived from the antagonistic HO-1 strain were sufficient to reproduce the antagonistic behavior and the efficiency of this inhibition correlated strongly with the HA content of the supernatant. Furthermore, reintroducing the capacity to produce HA to the 2-29 strain or supplementing bacterial co-cultures with HA restored antagonistic behavior. The HA-mediated antagonism was dose-dependent and affected by the temperature, but not by pH. HA caused a decline in biomass, cell aggregation, and hydrolysis of the cell wall in exponentially growing B. velezensis bulk cultures. Analysis of differential gene expression identified a series of genes modulating multicellular behavior in B. velezensis. Genes involved in motility, chemotaxis, sporulation, polypeptide synthesis, and non-ribosomal peptide synthesis were all significantly downregulated in the presence of HA, whereas autolysis-related genes showed upregulation. Taken together, these findings indicate that HA affects the population response of coexisting strains and also suggest that A. ammonioxydans HO-1 antagonize other bacteria by producing extracellular HA that, in turn, acts as a signaling molecule.

Degrading Characterization of the Newly Isolated Nocardioides sp. N39 for 3-Amino-5-methyl-isoxazole and the Related Genomic Information.

3-amino-5-methyl-isoxazole (3A5MI) is a persistent and harmful intermediate in the degradation of antibiotic sulfamethoxazole. It was accumulated in the environments day by day and has caused great environmental risks due to its refractory characteristic. Microbial degradation is economic and environmentally friendly and a promising method to eliminate this pollutant. In this study, a bacterial strain, Nocardioides sp. N39, was isolated. N39 can grow on 3A5MI as the sole carbon, nitrogen and energy resource. The effect of different factors on 3A5MI degradation by N39 was explored, including initial 3A5MI concentration, temperature, pH value, dissolved oxygen and additional carbon or nitrogen source. The degradation ability of N39 to various 3A5MI analogs was also explored. Nevertheless, the degrading ability of N39 for 3A5MI is not permanent, and long-term storage would lead to the loss of this ability. This may result from the mobile genetic elements in the bacterium according to the genomic comparison of N39 and its degrading ability-lost strain, N40. Despite this, N39 could support a lot of useful information about the degradation of 3A5MI and highlight the importance of studies about the environmental effects and potential degradation mechanism.

Dirammox Is Widely Distributed and Dependently Evolved in Alcaligenes and Is Important to Nitrogen Cycle.

Nitrogen cycle is an essential process for environmental health. Dirammox (direct ammonia oxidation), encoded by the dnfT1RT2ABCD cluster, was a novel pathway for microbial N2 production defined in Alcaligenes ammonioxydans HO-1. Here, a copy of the cluster dnfT1RT2ABCD as a whole was proved to have existed and very conserved in all Alcaligenes genomes. Phylogenetic analyses based on 16S rRNA gene sequences and amino acid sequences of DnfAs, together with G + C content data, revealed that dnf cluster was evolved associated with the members of the genus Alcaligenes. Under 20% O2 conditions, 14 of 16 Alcaligenes strains showed Dirammox activity, which seemed likely taxon-related. However, the in vitro activities of DnfAs catalyzing the direct oxidation of hydroxylamine to N2 were not taxon-related but depended on the contents of Fe and Mn ions. The results indicated that DnfA is necessary but not sufficient for Dirammox activity. The fact that members of the genus Alcaligenes are widely distributed in various environments, including soil, water bodies (both freshwater and seawater), sediments, activated sludge, and animal-plant-associated environments, strongly suggests that Dirammox is important to the nitrogen cycle. In addition, Alcaligenes species are also commonly found in wastewater treatment plants, suggesting that they might be valuable resources for wastewater treatment.

Degradation of amoxicillin by newly isolated Bosea sp. Ads-6.

Amoxicillin (AMX), one of the micro-amount hazardous pollutants, was frequently detected in environments, and of great risks to environments and human health. Microbial degradation is a promising method to eliminate pollutants. In this study, an efficient AMX-degrading strain, Ads-6, was isolated and characterized. Strain Ads-6, belonging to the genus Bosea, was also able to grow on AMX as the sole carbon and nitrogen source, with a removal of ~60% TOC. Ads-6 exhibited strong AMX-degrading ability at initial concentrations of 0.5-2 mM and pH 6-8. Addition of yeast extract could significantly enhance its degrading ability. Many degradation intermediates were identified by HPLC-MS, including new ones such as two phosphorylated products which were firstly defined in AMX degradation. A new AMX degradation pathway was proposed accordingly. Moreover, the results of comparative transcriptomes and proteomes revealed that ß-lactamase, L, D-transpeptidase or its homologous enzymes were responsible for the initial degradation of AMX. Protocatechuate branch of the beta-ketoadipate pathway was confirmed as the downstream degradation pathway. These results in the study suggested that Ads-6 is great potential in biodegradation of antibiotics as well as in the bioremediation of contaminated environments.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28 °C.

BACKGROUND: Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17-21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated. RESULTS: MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. CONTENT: Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. CONCLUSIONS: Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 µM, respectively. On the other hand, EB (800 µM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 µM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 µM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 µM) or TTX (2 µM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

Hydroxylation at Multiple Positions Initiated the Biodegradation of Indeno[1,2,3-cd]Pyrene in Rhodococcus aetherivorans IcdP1.

Nowadays, contamination by polycyclic aromatic hydrocarbons (PAHs) has become a serious problem all over the world; in particular, high-molecular-weight PAHs (HWM PAHs, four to seven rings) are more harmful to human health and environment due to their more complex structure and metabolic pathway. Biodegradation of PAHs with six or more rings, such as indeno[1,2,3-cd]pyrene (IcdP), was rarely described. An IcdP-degrading strain, Rhodococcus aetherivorans IcdP1, was isolated from HWM PAH-contaminated soil. It could grow on and efficiently degrade various HWM PAHs, such as IcdP, benzo[a]pyrene, and benzo[j]fluoranthene. It showed highest degrading ability toward IcdP (> 70% within 10 days). The IcdP degradation was initiated by ring hydroxylation with multiple pathways, including the hydroxylation at the 1,2 and 7,8 positions, according to the relevant metabolites detected, e.g., cyclopenta[cd]pyrene-3,4-dicarboxylic acid and 2,3-dimethoxy-2,3-dihydrofluoranthene. The transcriptional patterns of the genes encoding ring-hydroxylating oxygenases (RHOs) and cytochrome P450 monooxygenases (CYP450s) under the induction of IcdP, pyrene, and benzo[b]fluoranthene (BbF) were compared to determine the key initial RHOs in the conversion of IcdP. The expression of genes encoding RHOs 1892-1894, 1917-1920, and 4740-4741 was induced strictly by IcdP, and the amino acid sequences of these proteins showed very low identities with their homologs. These results suggested that IcdP was degraded through a dioxygenation-initiated metabolism pattern, and RHOs 1892-1894, 1917-1920, and 4740-4741 responded to the initial ring cleavage of IcdP through 1,2-dihydrodiol or 7,8-dihydrodiol. The studies would contribute to the understanding of the molecular mechanism of initial degradation of IcdP.

Curcumin, a Multi-Ion Channel Blocker That Preferentially Blocks Late Na+ Current and Prevents I/R-Induced Arrhythmias.

Increasing evidence shows that Curcumin (Cur) has a protective effect against cardiovascular diseases. However, the role of Cur in the electrophysiology of cardiomyocytes is currently not entirely understood. Therefore, the present study was conducted to investigate the effects of Cur on the action potential and transmembrane ion currents in rabbit ventricular myocytes to explore its antiarrhythmic property. The whole-cell patch clamp was used to record the action potential and ion currents, while the multichannel acquisition and analysis system was used to synchronously record the electrocardiogram and monophasic action potential. The results showed that 30 µmol/L Cur shortened the 50 and 90% repolarization of action potential by 17 and 7%, respectively. In addition, Cur concentration dependently inhibited the Late-sodium current (I Na.L), Transient-sodium current (I Na.T), L-type calcium current (I Ca.L), and Rapidly delayed rectifying potassium current (I Kr), with IC50 values of 7.53, 398.88, 16.66, and 9.96 µmol/L, respectively. Importantly, the inhibitory effect of Cur on I Na.L was 52.97-fold higher than that of I Na.T. Moreover, Cur decreased ATX II-prolonged APD, suppressed the ATX II-induced early afterdepolarization (EAD) and Ca2+-induced delayed afterdepolarization (DAD) in ventricular myocytes, and reduced the occurrence and average duration of ventricular tachycardias and ventricular fibrillations induced by ischemia-reperfusion injury. In conclusion, Cur inhibited I Na.L, I Na.T, I Ca.L, and I Kr; shortened APD; significantly suppressed EAD and DAD-like arrhythmogenic activities at the cellular level; and exhibited antiarrhythmic effect at the organ level. It is first revealed that Cur is a multi-ion channel blocker that preferentially blocks I Na.L and may have potential antiarrhythmic property.

Pseudomonas sp. AOB-7 utilizes PHA granules as a sustained-release carbon source and biofilm carrier for aerobic denitrification of aquaculture water.

Nitrate accumulation causes long-time threat to aquatic animals in recirculating aquaculture system (RAS); thus, nitrate removal is also required in RASs. However, the lack of carbon sources makes denitrification difficult to function. Nitrate removal performance of an aerobic denitrifying and extracellular polyhydroxyalkanoate depolymerase-producing bacterium, Pseudomonas sp. AOB-7, using polyhydroxyalkanoate (PHA) granules as a solid sustained-release carbon source in RAS was evaluated. With the initial nitrate-N concentration of 140 mg/L, the high denitrification rates of 0.056 g NO3--N L-1 day-1 and 0.035 g NO3--N L-1 day-1 were achieved in denitrification medium containing poly-ß-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), respectively. Significant erosions and pits formed on the surface of the granules made them a good biofilm carrier for AOB-7, and 3-hydroxybutyrate (3-HB) monomer was the major product released to aquatic phase, which was benefit to animals. SEM photos showed that AOB-7 entered and attached on the inside of the PHA particle holes. A 4-week application trial was conducted to reveal the effects of PHB (AOB-7) denitrifying agent and 3-HB produced on growth of zebrafish (Brachydanio rerio) by adding 0.1% (w/v) PHB (AOB-7) denitrifying agent. Result indicated that PHB (AOB-7) denitrifying agent can significantly reduce nitrate-N content in RASs. Compared with the control group, feed coefficient ratio reduced by 18% and weight gain ratio increased by 29% in the PHB (AOB-7) denitrifying agent group. 3-HB monomer produced during the denitrification was speculated to function as a prebiotic and promote zebrafish growth. KEY POINTS: • AOB-7 showed a good aerobic denitrifying ability on PHA granules as sustained-release C source. • PHB (AOB-7) denitrifying agent can significantly reduce nitrate content in RAS. • R-3-HB monomer was the major product released to aquatic phase and function as a prebiotic.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

Pseudomonas nitrititolerans sp. nov., a nitrite-tolerant denitrifying bacterium isolated from a nitrification/denitrification bioreactor.

A nitrite-tolerant denitrifying bacterium, strain GL14T, was isolated from the nitrification/denitrification bioreactor in our laboratory. Strain GL14T was Gram-stain-negative, rod-shaped, non-spore-forming, facultatively anaerobic and motile by means of a single polar flagellum. Phylogenetic analyses based on 16S rRNA gene sequences indicated that it was assigned to the genus Pseudomonas with highest 16S rRNA gene sequence similarity (98.77 %) to Pseudomonas xanthomarina DSM 18231T and Pseudomonassongnenensis NEAU-ST5-5T, followed by Pseudomonasstutzeri ATCC 17588T (98.42 %), Pseudomonaskunmingensis HL22-2T (98.29 %) and Pseudomonaszhaodongensis NEAU-ST5-21T (98.22 %). Phylogenetic analysis based on both concatenated sequences of the 16S rRNA gene and two housekeeping genes (gyrB and rpoD) and genome sequences further clarified the intrageneric phylogenetic position of strain GL14T. The DNA G+C content of GL14T was 63.1 mol%. The results of digital DNA-DNA hybridization (highest 24.2 % of DNA-DNA relatedness) based on the Genome-to-Genome Distance Calculator and average nucleotide identity analyses (highest 80.23 %) confirmed that the strain was distinctly delineated from known species of the genus Pseudomonas. The major fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C17 : 0cyclo and C12 : 0. The respiratory quinone was ubiquinone Q-9. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on the phylogenetic, genomic, phenotypic and chemotaxonomic analyses, it was concluded that strain GL14T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nitrititolerans sp. nov. is proposed. The type strain is GL14T (=CGMCC 1.13874T=NBRC 113853T).

Photobacterium sp. NNA4, an efficient hydroxylamine-transforming heterotrophic nitrifier/aerobic denitrifier.

An efficient heterotrophic nitrifying/aerobic denitrifying strain, Photobacterium sp. NNA4 was isolated from a recirculating aquaculture system (RAS). NNA4 was capable of utilizing ammonia, nitrate or nitrite as sole N-source with maximal removal rates of 12.5 mg/L/h for NH4+N, 16.4 mg/L/h for NO3--N, and 4.5 mg/L/h for NO2--N, respectively. Optimal nitrification conditions were: sodium succinate as C-source, 30-37°C, NaCl 1-4%, pH 7.0-8.0, dissolved oxygen 5.89 mg/L, C/N > 10. Gas chromatography/mass spectrometry and gas chromatography/isotope ratio mass spectrometry analyses showed that N2 and N2O were aerobic denitrification products of nitrite and nitrate. NNA4 could tolerate high concentration of hydroxylamine and displayed efficient hydroxylamine-transforming capability. Hydroxylamine oxidoreductase activity using potassium ferricyanide as electron acceptor was 0.042 U. Results revealed that strain NNA4 could oxidize NH2OH directly to N2O at aerobic conditions. In view of its high removal ability of inorganic nitrogen pollutants and broad salinity tolerance range, NNA4 has great potential in denitrification treatment of types of wastewater with either low salinity (e.g., municipal facilities) or high salinity (e.g., aquaculture, seafood processing).

Cross Talk between Chemosensory Pathways That Modulate Chemotaxis and Biofilm Formation.

Complex chemosensory systems control multiple biological functions in bacteria, such as chemotaxis, gene regulation, and cell cycle progression. Many species contain more than one chemosensory system per genome, but little is known about their potential interplay. In this study, we reveal cross talk between two chemosensory pathways that modulate chemotaxis and biofilm formation in Comamonas testosteroni We demonstrate that some chemoreceptors that govern chemotaxis also contribute to biofilm formation and these chemoreceptors can physically interact with components of both pathways. Finally, we show that the chemotaxis histidine kinase CheA can phosphorylate not only its cognate response regulator CheY2 but also one of the response regulators from the pathway mediating biofilm formation, FlmD. The phosphoryl group transfer from CheA to CheY2 is much faster than that from CheA to FlmD, which is consistent with chemotaxis being a fast response and biofilm formation being a much slower developmental process. We propose that cross talk between chemosensory pathways may play a role in coordination of complex behaviors in bacteria.IMPORTANCE In many bacteria, two or more homologous chemosensory pathways control several cellular functions, such as motility and gene regulation, in response to changes in the cell's microenvironment. Cross talk between signal transduction systems is poorly understood; while generally it is considered to be undesired, in some instances it might be beneficial for coregulation of complex behaviors. We demonstrate that several receptors from the pathway controlling motility can physically interact with downstream components of the pathway controlling biofilm formation. We further show that a kinase from the pathway controlling motility can also phosphorylate a response regulator from the pathway controlling biofilm formation. We propose that cross talk between two chemosensory pathways might be involved in coordination of two types of cell behavior-chemotaxis and biofilm formation.