Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue.

We previously demonstrated that Alcaligenes-derived lipid A (ALA), which is produced from an intestinal lymphoid tissue-resident commensal bacterium, is an effective adjuvant for inducing antigen-specific immune responses. To understand the immunologic characteristics of ALA as a vaccine adjuvant, we here compared the adjuvant activity of ALA with that of a licensed adjuvant (monophosphoryl lipid A, MPLA) in mice. Although the adjuvant activity of ALA was only slightly greater than that of MPLA for subcutaneous immunization, ALA induced significantly greater IgA antibody production than did MPLA during nasal immunization. Regarding the underlying mechanism, ALA increased and activated CD11b+ CD103- CD11c+ dendritic cells in the nasal tissue by stimulating chemokine responses. These findings revealed the superiority of ALA as a mucosal adjuvant due to the unique immunologic functions of ALA in nasal tissue.

Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO-1 to deal with the toxicity of ammonium.

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2 OH → N2 , first defined in Alcaligenes ammonioxydans HO-1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO-1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO-1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO-1 was revealed. After the disruption of the Dirammox in HO-1, more consumed NH4 + was recovered as biomass-N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia-rich wastewater treatment.

Dirammox-dominated microbial community for biological nitrogen removal from wastewater.

Direct ammonia oxidation (Dirammox) might be of great significance to advance the innovation of biological nitrogen removal process in wastewater treatment systems. However, it remains unknown whether Dirammox bacteria can be selectively enriched in activated sludge. In this study, a lab-scale bioreactor was established and operated for 2 months to treat synthetic wastewater with hydroxylamine as a selection pressure. Three Dirammox strains (Alcaligenes aquatilis SDU_AA1, Alcaligenes aquatilis SDU_AA2, and Alcaligenes sp. SDU_A2) were isolated from the activated sludge, and their capability to perform Dirammox process was confirmed. Although these three Dirammox bacteria were undetectable in the seed sludge (0%), their relative abundances rapidly increased after a month of operation, reaching 12.65%, 0.69%, and 0.69% for SDU_A2, SDU_AA1, and SDU_AA2, respectively. Among them, the most dominant Dirammox (SDU_A2) exhibited higher nitrogen removal rate (32.35%) than the other two strains (13.57% of SDU_AA1 and 14.52% of SDU_AA2). Comparative genomic analysis demonstrated that the most dominant Dirammox bacterium (SDU_A2) possesses fewer complete metabolic modules compared to the other two less abundant Alcaligenes strains. Our findings expanded the understanding of the application of Dirammox bacteria as key functional microorganisms in a novel biological nitrogen and carbon removal process if they could be well stabilized. KEY POINTS: • Dirammox-dominated microbial community was enriched in activated sludge bioreactor. • The addition of hydroxylamine played a role in Dirammox enrichment. • Three Dirammox bacterial strains, including one novel species, were isolated.

Nanosponge hydrogel of octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate of Alcaligenes faecalis.

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate (ODHP) was extracted in a previous study from the culture broth of soil isolate Alcaligenes faecalis MT332429 and showed a promising antimycotic activity. This study was aimed to formulate ODHP loaded ß-cyclodextrins (CD) nanosponge (NS) hydrogel (HG) to control skin fungal ailments since nanosponges augment the retention of tested agents in the skin. Box-Behnken design was used to produce the optimized NS formulation, where entrapment efficiency percent (EE%), polydispersity index (PDI), and particle size (PS) were assigned as dependent parameters, while the independent process parameters were polyvinyl alcohol % (w/v %), polymer-linker ratio, homogenization time, and speed. The carbopol 940 hydrogel was then created by incorporating the nanosponges. The hydrogel fit Higuchi's kinetic release model the best, according to in vitro drug release. Stability and photodegradation studies revealed that the NS-HG remained stable under tested conditions. The formulation also showed higher in vitro antifungal activity against Candida albicans compared to the control fluconazole. In vivo study showed that ODHP-NS-HG increased survival rates, wound contraction, and healing of wound gap and inhibited the inflammation process compared to the other control groups. The histopathological examinations and Masson's trichrome staining showed improved healing and higher records of collagen deposition. Moreover, the permeability of ODHP-NS-HG was higher through rats' skin by 1.5-folds compared to the control isoconazole 1%. Therefore, based on these results, NS-HG formulation is a potential carrier for enhanced and improved topical delivery of ODHP. Our study is a pioneering research on the development of a formulation for ODHP produced naturally from soil bacteria. KEY POINTS: • Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate was successfully formulated as a nanosponge hydrogel and statistically optimized. • The new formula exhibited in vitro good stability, drug release, and higher antifungal activity against C. albicans as compared to the fluconazole. • Ex vivo showed enhanced skin permeability, and in vivo analysis showed high antifungal activity as evidenced by measurement of various biochemical parameters and histopathological examination.

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.

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.

Pseudomonas paralcaligenes sp. nov., isolated from a hospitalized patient.

A Gram-stain-negative, aerobic, rod-shaped, non-endospore-forming bacterium, designated as strain MRCP1333T, was isolated from a faecal sample from a hospital patient in Japan. MRCP1333T grew at temperatures of 15-40 °C (optimum 25-35 °C), with 1.0-3.0 % (w/v, 171-513 mM) NaCl [optimum 1-2 % (w/v), 171-342 mM], and at pH 6.0-9.5 (optimum pH 7.0-8.0). The results of phylogenetic analysis based on the sequences of the 16S rRNA gene and the 53 genes encoding the bacterial ribosome protein subunits indicated that MRCP1333T represented a member of the Pseudomonas aeruginosa group, most closely related to Pseudomonas alcaligenes. Whole-genome comparisons, using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that MRCP1333T represented a distinct species in the P. aeruginosa group. Phenotypic characterization tests demonstrated utilization by this strain of citrate, glycerol, and d-malic acid, the ability to reduce nitrite to nitrogen and the ability of this strain to grow in the presence of minocycline and tetrazolium blue, distinguishing this strain from P. alcaligenes and other closely related species of the P. aeruginosa group. The major fatty acids of MRCP1333T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 38.4 %), summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 21.1 %) and C16 : 0 (20.6 %). The DNA G+C content of MRCP1333T was 66.5 mol%. Genetic and phenotypic evidence indicated that MRCP1333T should be classified as representing a novel species, for which the name Pseudomonas paralcaligenes sp. nov. is proposed. The type strain is MRCP1333T (=LMG 32254T,=JCM 34250T).

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.

The Novel Monooxygenase Gene dipD in the dip Gene Cluster of Alcaligenes faecalis JQ135 Is Essential for the Initial Catabolism of Dipicolinic Acid.

Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.

PicR as a MarR Family Transcriptional Repressor Multiply Controls the Transcription of Picolinic Acid Degradation Gene Cluster pic in Alcaligenes faecalis JQ135.

Picolinic acid (PA) is a natural toxic pyridine derivative as well as an important intermediate used in the chemical industry. In a previous study, we identified a gene cluster, pic, that responsible for the catabolism of PA in Alcaligenes faecalis JQ135. However, the transcriptional regulation of the pic cluster remains known. This study showed that the entire pic cluster was composed of 17 genes and transcribed as four operons: picR, picCDEF, picB4B3B2B1, and picT1A1A2A3T2T3MN. Deletion of picR, encoding a putative MarR-type regulator, greatly shortened the lag phase of PA degradation. An electrophoretic mobility shift assay and DNase I footprinting showed that PicR has one binding site in the picR-picC intergenic region and two binding sites in the picB-picT1 intergenic region. The DNA sequences of the three binding sites have the palindromic characteristics of TCAG-N4-CTNN: the space consists of four nonspecific bases, and the four palindromic bases on the left and the first two palindromic bases on the right are strictly conserved, while the last two bases on the right vary among the three binding sites. An in vivo ß-galactosidase activity reporter assay indicated that 6-hydroxypicolinic acid but not PA acted as a ligand of PicR, preventing PicR from binding to promoter regions and thus derepressing the transcription of the pic cluster. This study revealed the negative transcriptional regulation mechanism of PA degradation by PicR in A. faecalis JQ135 and provides new insights into the structure and function of the MarR-type regulator. IMPORTANCE The pic gene cluster was found to be responsible for PA degradation and widely distributed in Alpha-, Beta-, and Gammaproteobacteria. Thus, it is very necessary to understand the regulation mechanism of the pic cluster in these strains. This study revealed that PicR binds to three sites of the promoter regions of the pic cluster to multiply regulate the transcription of the pic cluster, which enables A. faecalis JQ135 to efficiently utilize PA. Furthermore, the study also found a unique palindrome sequence for binding of the MarR-type regulator. This study enhanced our understanding of microbial catabolism of environmental toxic pyridine derivatives.

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.

The efficient biomineralization and adsorption of cadmium (Cd2+) using secretory organo-biominerals (SOBs) produced by screened Alcaligenes faecalis K2.

Cadmium-contaminated wastewater has attracted increasing concerns due to its non-biodegradable properties and high toxicity. To explore eco-friendly and economically feasible strategies, the screened Alcaligenes faecalis K2 were employed for the biomineralization and recovery of Cd2+ from wastewater while producing considerable secretory organo-biominerals (SOBs) as bioadsorbents. At 75 mg/L Cd2+ exposure, 85.5% of Cd2+ was removed by K2, 43.0% of which was fixed in the granular SOBs. SOBs were convenient for separating from the solution. The adsorption capacity of granular sorbent made from SOBs was verified to be greater than 77.1 mg/g. Practically, 89.5% of 75 mg/L of Cd2+ could be stably removed while ereK2 continuously generated SOBs in a moving-bed biofilm reactor (MBBR). To sum up, the production of bioadsorbents can be achieved by K2, while removing Cd with live microorganisms, which was conducive to making full use of materials and improving Cd removal efficiency.

Simultaneous removal characteristics of ammonium and phenol by Alcaligenes faecalis strain WY-01 with the addition of acetate.

In this study, simultaneous removal of ammonium plus phenol could be achieved by Alcaligenes faecalis strain WY-01 with the addition of acetate, although acetate delayed the phenol degradation, probably due to the delayed expression of phenol hydroxylase gene under the presence of acetate. Moreover, the successful expression of key enzyme genes in strain WY-01 provided some evidence to illustrate its metabolic pathways of ammonium and phenol under aerobic conditions. Furthermore, SEM was used to clarify the role of acetate in resisting phenol toxicity, and these results demonstrated that strain WY-01 has the ability to form cell flocs when sodium acetate is used as co-substrate for a high concentration of phenol, and these flocs could protect cells against the toxicity of phenol, further enhancing phenol degradation in a high concentration of phenol. All these will provide further insights into the efficacy of strain WY-01 for treating wastewater cocontaminated by ammonium and phenol.

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.

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.

Lipid A-Mediated Bacterial-Host Chemical Ecology: Synthetic Research of Bacterial Lipid As and Their Development as Adjuvants.

Gram-negative bacterial cell surface component lipopolysaccharide (LPS) and its active principle, lipid A, exhibit immunostimulatory effects and have the potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, LPS and lipid A must be structurally modified to minimize their toxic effects while maintaining their adjuvant effect for application as vaccine adjuvants. In the field of chemical ecology research, various biological phenomena occurring among organisms are considered molecular interactions. Recently, the hypothesis has been proposed that LPS and lipid A mediate bacterial-host chemical ecology to regulate various host biological phenomena, mainly immunity. Parasitic and symbiotic bacteria inhabiting the host are predicted to possess low-toxicity immunomodulators due to the chemical structural changes of their LPS caused by co-evolution with the host. Studies on the chemical synthesis and functional evaluation of their lipid As have been developed to test this hypothesis and to apply them to low-toxicity and safe adjuvants.

Potential application in amoxicillin removal of Alcaligenes sp. MMA and enzymatic studies through molecular docking.

Antibiotic contamination in environmental matrices is a serious global problem which leads to an increase in the proliferation of antibiotic resistance genes. Amoxicillin is ubiquitous in the environment, but there is hardly any information on the dissipation of amoxicillin by the microbial community. In view of this, the present study focusses on the removal of amoxicillin using amoxicillin-resistant bacteria, Alcaligenes sp. MMA. Bacteria were characterized using antibiotic tests, biochemical and molecular analysis. Alcaligenes sp. MMA was able to remove up to 84% of amoxicillin in 14 days in M9 minimal media, and the degradation products were confirmed using LC-MS/MS, including the benzothiazole, 2-Amino-3-methoxyl benzoic acid, 4-Hydroxy-2-methyl benzoic acid, 5-Amino-2-methylphenol and 3,5-Bis(tert-butyl)-2-hydroxybenzaldehyde, at the end of 14th day which further shows the removal of amoxicillin by the bacterial strain. Differential expression of porins was found in the presence of amoxicillin as a sole source of carbon and energy for the bacterial strain. Molecular interaction using in silico studies were performed which showed the formation of a hydrogen bond between amoxicillin and porins.

Co-expression of Pseudomonas alcaligenes lipase and its specific foldase in Pichia pastoris by a dual expression cassette strategy.

Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases.

Extensively drug-resistant Alcaligenes faecalis infection.

BACKGROUND: Alcaligenes faecalis is usually causes opportunistic infections in humans. Alcaligenes faecalis infection is often difficult to treat due to its increased resistance to several antibiotics. The results from a clinical study of patients with Alcaligenes faecalis infection may help improve patients' clinical care. METHODS: We conducted a retrospective analysis of all patients presenting with Alcaligenes faecalis infection from January 2014 to December 2019. The medical records of all patients were reviewed for demographic information, clinical symptoms and signs, comorbidities, use of intravenous antibiotics within the past three months, bacterial culture, antibiotics sensitivity test, and clinical outcomes. RESULTS: Sixty-one cases of Alcaligenes faecalis infection were seen during the study period, including 25 cases of cystitis, nine cases of diabetic foot infection, eight cases of pneumonia, seven cases of acute pyelonephritis, three cases of bacteremia, and nine cases of infection at specific sites. Thirty-seven patients (60.7%) had a history of receiving intravenous antibiotics within three months of the diagnosis. Fifty-one (83.6%) cases were mixed with other bacterial infections. Extensively drug-resistant infections have been reported since 2018. The best sensitivity rate to Alcaligenes faecalis was 66.7% for three antibiotics (imipenem, meropenem, and ceftazidime) in 2019. Two antibiotics (ciprofloxacin and piperacillin/tazobactam) sensitivity rates to A. faecalis were less than 50%. CONCLUSIONS: The most frequent Alcaligenes faecalis infection sites, in order, are the bloodstream, urinary tract, skin and soft tissue, and middle ear. The susceptibility rate of Alcaligenes faecalis to commonly used antibiotics is decreasing. Extensively drug-resistant Alcaligenes faecalis infections have emerged.