Hydroxylamine production by Alcaligenes faecalis challenges the paradigm of heterotrophic nitrification.

Heterotrophic nitrifiers continue to be a hiatus in our understanding of the nitrogen cycle. Despite their discovery over 50 years ago, the physiology and environmental role of this enigmatic group remain elusive. The current theory is that heterotrophic nitrifiers are capable of converting ammonia to hydroxylamine, nitrite, nitric oxide, nitrous oxide, and dinitrogen gas via the subsequent actions of nitrification and denitrification. In addition, it was recently suggested that dinitrogen gas may be formed directly from ammonium. Here, we combine complementary high-resolution gas profiles, 15N isotope labeling studies, and transcriptomics data to show that hydroxylamine is the major product of nitrification in Alcaligenes faecalis. We demonstrated that denitrification and direct ammonium oxidation to dinitrogen gas did not occur under the conditions tested. Our results indicate that A. faecalis is capable of hydroxylamine production from an organic intermediate. These results fundamentally change our understanding of heterotrophic nitrification and have important implications for its biotechnological application.

Bioelectricity facilitates carbon dioxide fixation by Alcaligenes faecalis ZS-1 in a biocathodic microbial fuel cell (MFC).

The CO2 fixation mechanism by Alcaligenes faecalis ZS-1 in a biocathode microbial fuel cell (MFC) was investigated. The closed-circuit MFC (CM) exhibited a significantly higher CO2 fixation rate (10.7%) compared to the open-circuit MFC (OC) (2.0%), indicating that bioelectricity enhances CO2 capture efficiency. During the inward extracellular electron transfer (EET) process, riboflavin concentration increased in the supernatant while cytochrome levels decreased. Genome sequencing revealed diverse metabolic pathways for CO2 fixation in strain ZS-1, with potential dominance of rTCA and C4 pathways under electrotrophic conditions as evidenced by significant upregulation of the ppc gene. Differential metabolite analysis using LC-MS demonstrated that CM promoted upregulation of various lipid metabolites. These findings collectively highlight that ZS-1 simultaneously generated electricity and fixed CO2 and that the ppc associated with bioelectricity played a critical role in CO2 capture. In conclusion, bioelectricity resulted in a significant enhancement in the efficiency of CO2 fixation and lipid production.

Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium.

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.

Functional genomic analysis of an efficient indole degrading bacteria strain Alcaligenes faecalis IITR89 and its biodegradation characteristics.

Indole is a nitrogenous heterocyclic aromatic pollutant often detected in various environments. An efficient indole degrading bacterium strain IITR89 was isolated from River Cauvery, India, and identified as Alcaligenes faecalis subsp. phenolicus. The bacterium was found to degrade ~ 95% of 2.5 mM (293.75 mg/L) of indole within 18 h utilizing it as a sole carbon and energy source. Based on metabolite identification, the metabolic route of indole degradation is indole → (indoxyl) → isatin → (anthranilate) → salicylic acid → (catechol) → (Acetyl-CoA) → and further entering into TCA cycle. Genome sequencing of IITR89 revealed the presence of gene cluster dmpKLMNOP, encoding multicomponent phenol hydroxylase; andAbcd gene cluster, encoding anthranilate 1,2-dioxygenase ferredoxin subunit (andAb), anthranilate 1,2-dioxygenase large subunit (andAc), and anthranilate 1,2-dioxygenase small subunit (andAd); nahG, salicylate hydroxylase; catA, catechol 1,2-dioxygenase; catB, cis, cis-muconate cycloisomerase; and catC, muconolactone D-isomerase which play an active role in indole degradation. The findings strongly support the degradation potential of strain IITR89 and its possible application for indole biodegradation.

Phenotypic and genomic characterization provide new insights into adaptation to environmental stressors and biotechnological relevance of mangrove Alcaligenes faecalis D334.

Alcaligenes faecalis D334 was determined in this study as a salt-tolerant bacterium isolated from mangrove sediment. In response to 6% (w/v) NaCl, strain D334 produced the highest ectoines of 14.14 wt%. To understand adaptive features to mangrove environment, strain D334 was sequenced using Pacific BioScience platform, resulting in a circular chromosome of 4.23 Mb. Of note, D334 genome harbored 81 salt-responsive genes, among which two membrane-associated genes ompc and eric were absent in 3 selected A. faecalis genomes. Apart from that, a complete pathway for ectoine and 5-hydroxyectoine synthesis was predicted. To resist 40 mM H2O2, 46 genetic determinants contributing to oxidative stress response were employed. Moreover, two operons involved in polyhydroxyalkanoate (PHA) production were identified in the D334 genome, resulting in maximum PHA content of 5.03 ± 0.04 wt% and PHA concentration of 0.13 ± 0.001 g/L. A large flagellar biosynthesis operon contributing to swimming motility was found to be conserved in D334 and 8 other A. faecalis genomes. These findings shed light for the first time on the high versatility of A. faecalis D334 genome to adapt to mangrove lifestyle and the possibility to develop D334 as an industrial platform for PHA and 5-hydroxyectoine production.

The MocR family transcriptional regulator DnfR has multiple binding sites and regulates Dirammox gene transcription in Alcaligenes faecalis JQ135.

Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3 →NH2 OH→N2 ), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR-like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506-bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated conserved sequence 5'-GGTCTG-N17 -GGTCTG-3' which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.

Quinolinic acid catabolism is initiated by a novel four-component hydroxylase QuiA in Alcaligenes faecalis JQ191.

Quinolinic acid (QA) is an essential nitrogen-containing aromatic heterocyclic compounds in organisms and it also acts as an important intermediate in chemical industry, which has strong neurotoxicity and cytotoxicity. The wide range of sources and applications caused the release and accumulation of QA in the environment which might poses a hazard to ecosystems and human health. However, few research on the degradation of QA by microorganisms and toxicity of QA and its metabolites were reported. Alcaligenes faecalis JQ191 could degrade QA but the genetic foundation of QA degradation has not been studied. In this study, the gene cluster quiA1A2A3A4 was identified from A. faecalis JQ191, which was responsible for the initial catabolism step of QA. The quiA1A2A3A4 gene cluster encodes a novel cytoplasmic four-component hydroxylase QuiA. The 1H nuclear magnetic resonance indicated that QuiA catalyzed QA to 6-hydroxyquinolinic acid (6HQA) and the H218O-labeling analysis confirmed that the hydroxyl group incorporating into 6HQA was derived from water. Toxicity tests showed that the QA could approximately inhibit 20%-80% growth of Chlorella ellipsoidea, and 6HQA could relieve at least 50% QA growth inhibition of Chlorella ellipsoidea, indicating that the 6-hydroxylation of QA by QuiA is a detoxification process. This research provides new insights into the metabolism of QA by microorganism and potential application in the bioremediation of toxic pyridine derivatives-contaminated environments.

Nitrogen removal and mechanism of an extremely high-ammonia tolerant heterotrophic nitrification-aerobic denitrification bacterium Alcaligenes faecalis TF-1.

A novel heterotrophic nitrifying bacterium with high salt and high ammonia nitrogen tolerance, Alcaligenes faecalis TF-1, was isolated from the leachate of a landfill. The verification of nitrogen removal efficiency of different nitrogen sources and PCR amplification electrophoresis results showed that the HN-AD pathway of the strain TF-1 was NH4+ â†’ NH2OH â†’ NO â†’ N2O â†’ N2. The results of parameter optimization showed that the optimal nitrogen removal conditions were as follows: sodium citrate as carbon source, C/N = 16, pH = 7, and NH4+-N loading of 808.21 mg/L. The strain TF-1 could remove about 94.60% of ammonia nitrogen (1963.94 mg/L). The salinity tolerance range of the strain TF-1 was 0-70 g/L, and the removal efficiency was 52.87% at salinity 70 g/L and NH4+-N concentration 919.20 mg/L and 55.67% at pH = 10 and NH4+-N concentration 994.82 mg/L. The extreme environmental adaptability and remarkable nitrogen removal performance make this strain a promising candidate in leachate treatment.

Genomic and resistome analysis of Alcaligenes faecalis strain PGB1 by Nanopore MinION and Illumina Technologies.

BACKGROUND: Drug-resistant bacteria are important carriers of antibiotic-resistant genes (ARGs). This fact is crucial for the development of precise clinical drug treatment strategies. Long-read sequencing platforms such as the Oxford Nanopore sequencer can improve genome assembly efficiency particularly when they are combined with short-read sequencing data. RESULTS: Alcaligenes faecalis PGB1 was isolated and identified with resistance to penicillin and three other antibiotics. After being sequenced by Nanopore MinION and Illumina sequencer, its entire genome was hybrid-assembled. One chromosome and one plasmid was assembled and annotated with 4,433 genes (including 91 RNA genes). Function annotation and comparison between strains were performed. A phylogenetic analysis revealed that it was closest to A. faecalis ZD02. Resistome related sequences was explored, including ARGs, Insert sequence, phage. Two plasmid aminoglycoside genes were determined to be acquired ARGs. The main ARG category was antibiotic efflux resistance and ß-lactamase (EC 3.5.2.6) of PGB1 was assigned to Class A, Subclass A1b, and Cluster LSBL3. CONCLUSIONS: The present study identified the newly isolated bacterium A. faecalis PGB1 and systematically annotated its genome sequence and ARGs.

Biodegradation of quinolinic acid by a newly isolated bacterium Alcaligenes faecalis strain JQ191.

Quinolinic acid (QA) is a pyridine derivative that can be found in many organisms and is widely used in the chemical industry. However, QA possesses excitotoxic properties. To date, the catabolism of QA mediated by microorganisms has rarely been reported. In this study, a QA-degrading strain (JQ191) was isolated from sewage sludge. Based on phenotypic and 16S rRNA gene phylogenetic analysis, the strain was identified as Alcaligenes faecalis. Strain JQ191 was able to utilize QA as the sole source of carbon and nitrogen for growth. QA-cultured cells of JQ191 completely degrade 200 mg/L QA within 2 days in a mineral salt medium, whereas the LB-cultured cells experienced a 2-day lag period before degrading QA, indicating that the catabolic enzymes involved in QA degradation were induced by QA. 6-Hydroxypicolinic acid (6HPA) was identified as an intermediate of QA degradation by strain JQ191. A 6HPA monooxygenase gene picB was cloned, genetically disrupted, and heterologously expressed, and the results show that picB was responsible for catalyzing 6HPA to 3,6DHPA in JQ191. A new QA mineralization pathway was proposed. This study identifies a new bacterium candidate that has a potential application prospect in the bioremediation of QA-polluted environment, as well as provides new insights into the bacterial catabolism of QA.

The TetR Family Repressor HpaR Negatively Regulates the Catabolism of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135 by Binding to Two Unique DNA Sequences in the Promoter of Hpa Operon.

5-Hydroxypicolinic acid (5HPA), an important natural pyridine derivative, is microbially degraded in the environment. Previously, a gene cluster, hpa, responsible for 5HPA degradation, was identified in Alcaligenes faecalis JQ135. However, the transcription regulation mechanism of the hpa cluster is still unknown. In this study, the transcription start site and promoter of the hpa operon was identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR, whereas the transcription of hpaR itself was not regulated by HpaR. Electrophoretic mobility shift assay and DNase I footprinting revealed that HpaR bound to two DNA sequences, covering the -35 region and -10 region, respectively, in the promoter region of the hpa operon. Interestingly, the two binding sequences are partially palindromic, with 3 to 4 mismatches and are complementary to each other. 5HPA acted as a ligand of HpaR, preventing HpaR from binding to promoter region and derepressing the transcription of the hpa operon. The study revealed that HpaR binds to two unique complementary sequences of the promoter of the hpa operon to negatively regulate the catabolism of 5HPA. IMPORTANCE This study revealed that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR. The binding of HpaR to the promoter of the hpa operon has the following unique features: (i) HpaR has two independent binding sites in the promoter of the hpa operon, covering -35 region and -10 region, respectively; (ii) the palindrome sequences of the two binding sites are complementary to each other; and (iii) both of the binding sites include a 10-nucleotide partial palindrome sequence with 3 to 4 mismatches. This study provides new insights into the binding features of the TetR family regulator with DNA sequences.

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.

Complete genome analysis of the novel Alcaligenes faecalis phage vB_AfaP_QDWS595.

A novel lytic phage named vB_AfaP_QDWS595 infecting Alcaligenes faecalis was isolated and characterized in this study. The genome of phage vB_AfaP_QDWS595 was sequenced and analyzed, and the result revealed that the phage contained 70,466 bp of double-stranded DNA with 41.12% GC content. There were 74 putative genes encoding proteins as well as 11 tRNAs predicted in the phage genome. Phenotype and phylogeny analysis indicated that this phage might be a new member of the family Schitoviridae.

Constitutive secretory expression and characterization of nitrilase from Alcaligenes faecalis in Pichia pastoris for production of R-mandelic acid.

Nitrilases can directly hydrolyze nitrile compounds into carboxylic acids and ammonium. To solve the current problems of bioconversions using nitrilases, including the difficult separation of products from the resting cells used as the catalyst and high costs of chemical inducers, a nitrilase from Alcaligenes faecalis was heterologously expressed in Pichia pastoris X33. The stable nitrilase-expressing strain No.39-6-4 was obtained after three rounds of screening based on a combined detection method including dot-blot, SDS-PAGE, and western blot analyses, which confirmed the presence of recombinant nitrilase with a molecular mass of about 50 kDa. The temperature and pH optima of the nitrilase were 45°C and pH 7.5, respectively. Cu2+ , Zn2+ , and Tween 80 strongly inhibited the enzyme activity, but the optical purity of the product R-mandelic acid (R-MA) was stable, with practically 100% enantiomeric excess (ee). The nitrilase-producing P. pastoris strain developed in this study provides a basis for further research on the enzyme.

Alcaligenes faecalis metallo-ß-lactamase in extensively drug-resistant Pseudomonas aeruginosa isolates.

OBJECTIVES: To characterize Alcaligenes faecalis metallo-ß-lactamase (MBL) AFM-2 and AFM-3 from clinical Pseudomonas aeruginosa isolates NDTH10366, NDTH9845 and WTJH17. METHODS: Clinical isolates were whole-genome sequenced using the Illumina and Oxford Nanopore platforms. MICs of clinical isolates and transformants containing MBL genes were determined using broth microdilution methods. Kinetic parameters of purified AFM and NDM-1 were measured using a spectrophotometer. The AFM structure was modelled with SWISS-MODEL. RESULTS: NDTH10366 and NDTH9845 were extensively drug-resistant (XDR) isolates carrying blaAFM-2 and multiple copies of blaKPC-2, whereas WTJH17 was an XDR isolate carrying blaAFM-3. The plasmid-borne blaAFM-2 and blaAFM-3 genes are associated with a novel ISCR element, ISCR29. AFM-2 and AFM-3, differing from AFM-1 by one amino acid substitution each, shared 86.2% and 86.6% amino acid sequence identity with NDM-1, respectively. Phylogenetic analysis confirmed the close relationship between AFM and NDM. Expression of AFM and NDM-1 under their native promoters in DH5α and PAO1 led to elevated MICs for all tested ß-lactams except aztreonam. Comparable catalytic abilities were observed for AFM and NDM-1 when hydrolysing nitrocefin, cefepime, imipenem and biapenem, whereas for other tested ß-lactams AFM displayed weaker enzymatic activities. Modelling AFM structure revealed a characteristic αß/ßα fold with two zinc-binding active sites. CONCLUSIONS: AFM from clinical P. aeruginosa isolates demonstrated ß-lactamase activity comparable to NDM-1. Co-carriage of blaAFM and blaKPC renders clinical P. aeruginosa isolates non-susceptible to all antipseudomonal ß-lactams. The association of blaAFM genes with translocatable genetic elements and plasmids highlights their concerning potential for dissemination.

Exploring new marine bacterial species, Alcaligenes faecalis Alca F2018 valued for bioconversion of shrimp chitin to chitosan for concomitant biotechnological applications.

The exploitation of chitinous materials seems to be an infinite treasure. To this end, using shellfish waste as the sole carbon/nitrogen source solves environmental challenges while lowering microbial chitinase production costs. Bioconversion of shellfish chitin wastes such as shrimp shells has recently been investigated for the production of enzymes and bioactive materials in order to maximize the utilization of chitin-containing seafood processing wastes. In this study, the bioconversion of chitin to chitosan by Alcaligenes faecalis Alca F2018 revealed the highest chitin deacetylase (CDA) activity of 40.6 U/µg. The resulted low Km and high Vmax values explain the high affinity of the purified CDA to the p-nitroacetanilide substrate. CDA with a molecular weight of 66 KDa was purified from F2018 strain, with a 14.5% yield. FT-IR revealed distinct chitosan peaks and XRD revealed that chitosan samples had lower crystallinity than chitin. TGA analysis revealed that the recovered chitosan samples were more thermally stable. The deacetylation degree percentages of the produced chitosan are in the same range as that of the commercial chitosan, suggesting the promising potential of A. faecalis Alca F2018 to utilize shrimp shells in their raw form in the fermentation media based on its CDA enzyme activity.

Gene expression analysis of Alcaligenes faecalis during induction of heterotrophic nitrification.

Alcaligenes faecalis is a heterotrophic nitrifying bacterium that oxidizes ammonia and generates nitrite and nitrate. When A. faecalis was cultivated in a medium containing pyruvate and ammonia as the sole carbon and nitrogen sources, respectively, high concentrations of nitrite accumulated in the medium whose carbon/nitrogen (C/N) ratio was lower than 10 during the exponential growth phase, while the accumulation was not observed in the medium whose C/N ratio was higher than 15. Comparative transcriptome analysis was performed using nitrifying and non-nitrifying cells of A. faecalis cultivated in media whose C/N ratios were 5 and 20, respectively, to evaluate the fluctuations of gene expression during induction of heterotrophic nitrification. Expression levels of genes involved in primary metabolism did not change significantly in the cells at the exponential growth phase under both conditions. We observed a significant increase in the expression levels of four gene clusters: pod cluster containing the gene encoding pyruvic oxime dioxygenase (POD), podh cluster containing the gene encoding a POD homolog (PODh), suf cluster involved in an iron-sulfur cluster biogenesis, and dnf cluster involved in a novel hydroxylamine oxidation pathway in the nitrifying cells. Our results provide valuable insight into the biochemical mechanism of heterotrophic nitrification.

Heterotrophic nitrification and related functional gene expression characteristics of Alcaligenes faecalis SDU20 with the potential use in swine wastewater treatment.

A new heterotrophic nitrifying bacterium was isolated from the compost of swine manure and rice husk and identified as Alcaligenes faecalis SDU20. Strain SDU20 had heterotrophic nitrification potential and could remove 99.7% of the initial NH4+-N. Nitrogen balance analysis revealed that 15.9 and 12.3% of the NH4+-N were converted into biological nitrogen and nitrate nitrogen, respectively. The remaining 71.44% could be converted into N2 or N2O. Single-factor experiments showed that the optimal conditions for ammonium removal were the carbon source of sodium succinate, C/N ratio 10, initial pH 8.0, and temperature 30 °C. Nitrification genes were determined to be upregulated when sodium succinate was used as the carbon source analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Strain SDU20 could tolerate 4% salinity and show resistance to some heavy metal ions. Strain SDU20 removed 72.6% high concentrated NH4+-N of 2000 mg/L within 216 h. In a batch experiment, the highest NH4+-N removal efficiency of 98.7% and COD removal efficiency of 93.7% were obtained in the treatment of unsterilized swine wastewater. Strain SDU20 is promising in high-ammonium wastewater treatment.

Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue.

AfIP-1A/1B is a two-component insecticidal protein identified from the soil bacterium Alcaligenes faecalis that has high activity against western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). Previous results revealed that AfIP-1A/1B is cross-resistant to the binary protein from Bacillus thuringiensis (Bt), Cry34Ab1/Cry35Ab1 (also known as Gpp34Ab1/Tpp35Ab1; Crickmore et al., 2020), which was attributed to shared binding sites in WCR gut tissue (Yalpani et al., 2017). To better understand the interaction of AfIP-1A/1B with its receptor, we have systematically evaluated the binding of these proteins with WCR brush border membrane vesicles (BBMVs). Our findings show that AfIP-1A binds directly to BBMVs, while AfIP-1B does not; AfIP-1B binding only occurred in the presence of AfIP-1A which was accompanied by the presence of stable, high molecular weight oligomers of AfIP-1B observed on denaturing protein gels. Additionally, we show that AfIP-1A/1B forms pores in artificial lipid membranes. Finally, binding of AfIP-1A/1B was found to be reduced in BBMVs from Cry34Ab1/Cry35Ab1-resistant WCR where Cry34Ab1/Cry35Ab1 binding was also reduced. The reduced binding of both proteins is consistent with recognition of a shared receptor that has been altered in the resistant strain. The coordination of AfIP-1B binding by AfIP-1A, the similar structures between AfIP-1A and Cry34Ab1, along with their shared binding sites and cross-resistance, suggest a similar role for AfIP1A and Cry34Ab1 in receptor recognition and docking site for their cognate partners, AfIP-1B and Cry35Ab1, respectively.

Nitrogen removal characteristics of a novel heterotrophic nitrification and aerobic denitrification bacteria, Alcaligenes faecalis strain WT14.

Alcaligenes faecalis strain WT14 is heterotrophic nitrification and aerobic denitrification bacterium, newly isolated from a constructed wetland, and its feasibility in nitrogen removal was investigated. The result showed sodium citrate was more readily utilized by WT14 as a carbon source. The response surface methodology model revealed the highest total nitrogen removal by WT14 occurred at 20.3 °C, 113.5 r·min-1, C/N 10.8, and pH 8.4. Under adapted environmental conditions, up to 55.9 mg·L-1·h-1 of ammonium nitrogen (NH4+-N) was removed by WT14, and its NH4+-N tolerance ability reached 2000 mg·L-1. In addition to the reported high NH4+-resistance of Alcaligenes faecalis, WT14 multiplied fast and had strong nitrate or nitrite removal capacity when high strength nitrate or nitrite was provided as the single nitrogen source; which differed from other Alcaligenes faecalis species. These results show WT14 is a novel strain of Alcaligenes faecalis and its nitrogen removal pathway will be carried out in the further study.