In vitro and in vivo screening of bacterial species from contaminated soil for heavy metal biotransformation activity.

Heavy metals (HMs) are widely used in various industries. High concentrations of HMs can be severely toxic to plants, animals and humans. Microorganism-based bioremediation has shown significant potential in degrading and detoxifying specific HM contaminants. In this study, we cultivated a range of bacterial strains in liquid and solid nutrient medium containing different concentrations of different HMs to select and analyze bacteria capable of transforming HMs. The bacterial strains most resistant to selected HMs and exhibiting the ability to remove HMs from contaminated soils were identified. Then, the bacterial species capable of utilizing HMs in soil model experiments were selected, and their ability to transform HMs was evaluated. This study has also generated preliminary findings on the use of plants for further removal of HMs from soil after microbial bioremediation. Alcaligenes faecalis, Delftia tsuruhatensis and Stenotrophomonas sp. were selected for their ability to grow in and utilize HM ions at the maximum permissible concentration (MPC) and two times the MPC. Lysinibacillus fusiformis (local microflora) can be used as a universal biotransformation tool for many HM ions. Brevibacillus parabrevis has potential for the removal of lead ions, and Brevibacillus reuszeri and Bacillus safensis have potential for the removal of arsenic ions from the environment. The bacterial species have been selected for bioremediation to remove heavy metal ions from the environment.

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.

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.

Biodegradation of low-density polyethylene (LDPE) through application of indigenous strain Alcaligenes faecalis ISJ128.

The resiliency of plastic products against microbial degradation in natural environment often creates devastating changes for humans, plants, and animals on the earth's surface. Biodegradation of plastics using indigenous bacteria may serve as a critical approach to overcome this resulting environmental stress. In the present work, a polyethylene degrading bacterium Alcaligenes faecalis strain ISJ128 (Accession No. MK968769) was isolated from partially degraded polyethylene film buried in the soil at plastic waste disposal site. The biodegradation studies were conducted by employing various methods such as hydrophobicity assessment of the strain ISJ128, measurement of viability and total protein content of bacterial biofilm attached to the polyethylene surface. The proliferation of bacterial cells on polyethylene film, as indicated by high growth response in terms of protein content (85.50 µg mL-1) and viability (1010 CFU mL-1), proposed reasonable suitability of our strain A. faecalis ISJ128 toward polyethylene degradation. The results of biodegradation assay revealed significant degradation (10.40%) of polyethylene film within a short period of time (i.e., 60 days), whereas no signs of degradation were seen in control PE film. A. faecalis strain ISJ128 also demonstrated a removal rate of 0.0018 day-1 along with half-life of 462 days. The scanning electron microscope (SEM) and Fourier transform infrared (FTIR) spectroscopy studies not only displayed changes on polyethylene surface but also altered level of intensity of functional groups and an increase in the carbonyl indexes justifying the degradation of polyethylene film due to bacterial activity. In addition, the secondary structure prediction (M fold software) of 16SrDNA proved the stable nature of the bacterial strain, thereby reflecting the profound scope of A. faecalis strain ISJ128 as a potential degrader for the eco-friendly disposal of polyethylene waste. Schematic representation of methodology.

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.

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.

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.

Biocontrol activity and putative mechanism of Bacillus amyloliquefaciens (SF14 and SP10), Alcaligenes faecalis ACBC1, and Pantoea agglomerans ACBP1 against brown rot disease of fruit.

This study aimed at evaluating the antagonistic activity of 16 bacterial strains for the control of brown rot disease caused by Monilinia fructigena, and M. laxa under in vitro and a semi-commercial large-scale trial. These bacterial antagonists' belonging to the genera Alcaligenes, Bacillus, Brevibacterium, Pantoea, Pseudomonas, and Serratia were previously proven effective for control of fire blight of apple. The in vitro dual culture bioassay showed the highest inhibition rates of mycelial growth ranging from 55 to 95% and from 43 to 94% for M. fructigena and M. laxa, respectively. The in vivo bioassay showed moderate and strong inhibition for M. fructigena and M. laxa, respectively. The inhibition rates were dependent on incubation time as well as pathogen virulence. The free-cell bacterial filtrate revealed substantial mycelial growth inhibition ranging from 66 to 86%. The inhibition of conidial germination was from 32 to 78%, suggesting the involvement of metabolites in their biocontrol activity. The antifungal effect of the volatile compounds (VCOs) was observed for all bacteria with mycelial inhibition varying from 12 to 70%. Overall, their efficacy was substantially affected by the nature of the bacterial strains and the modes of action. Taken together, these results underscore that ACBC1 and SF14 for M. fructigena and SP10 and ACBP1 for M. laxa were the most effective bacterial strains. These strains were confirmed effective in a semi-commercial large-scale trial. Interestingly, their efficacies were found to be comparable to those of both commercial BCAs (B. subtilis Y1336 and P. agglomerans P10c), but slightly lower than thiophanate-methyl fungicide. The ability of most bacterial strains to produce lytic enzymes (Amylase, Protease or Cellulase) and lipopeptides (bacillomycin, fengycin, iturin and surfactin) was demonstrated by biochemical and molecular analyzes. Therefore, our findings suggest that the bacterial antagonists ACBC1, SF14, SP10 and ACBP1, have the potential to prevent brown rot disease.

Bioelectrochemical behaviour of a sequentially added biocatalytic coculture in a microbial fuel cell.

Bacterial isolates Pseudomonas aeruginosa BR, Alcaligenes faecalis SW and Escherichia coli EC from a microbial fuel cell (MFC) were cocultured with each other. The isolates were added in a specific sequence one after the other (two cultures in one reactor). The study helped us conceptualise the synergistic and antagonistic behaviour of bacterial isolates and also emphasised the sequence in which a culture can be introduced in an anode chamber. The coculture conditions were evaluated on the basis of redox activity, electron transfer rate, columbic efficiency, and internal resistances. A good syntropy in terms of power production was found between P. aeruginosa BR and E. coli EC that recorded a power generation of 207.593 ± 1.705 µW/m2 . The addition of any culture in any sequence affected the performance of P. aeruginosa BR. This culture when added as a secondary culture in the reactor enhanced the performance of that reactor. The sequence of addition of a culture in a reactor affected the performance in the combinations of (a) P. aeruginosa BR and A. faecalis SW and (b) A. faecalis SW and E. coli EC.

Identification and Characterization of a Novel pic Gene Cluster Responsible for Picolinic Acid Degradation in Alcaligenes faecalis JQ135.

Picolinic acid (PA) is a natural toxic pyridine derivative. Microorganisms can degrade and utilize PA for growth. However, the full catabolic pathway of PA and its physiological and genetic foundation remain unknown. In this study, we identified a gene cluster, designated picRCEDFB4B3B2B1A1A2A3, responsible for the degradation of PA from Alcaligenes faecalis JQ135. Our results suggest that PA degradation pathway occurs as follows: PA was initially 6-hydroxylated to 6-hydroxypicolinic acid (6HPA) by PicA (a PA dehydrogenase). 6HPA was then 3-hydroxylated by PicB, a four-component 6HPA monooxygenase, to form 3,6-dihydroxypicolinic acid (3,6DHPA), which was then converted into 2,5-dihydroxypyridine (2,5DHP) by the decarboxylase PicC. 2,5DHP was further degraded to fumaric acid through PicD (2,5DHP 5,6-dioxygenase), PicE (N-formylmaleamic acid deformylase), PicF (maleamic acid amidohydrolase), and PicG (maleic acid isomerase). Homologous pic gene clusters with diverse organizations were found to be widely distributed in Alpha-, Beta-, and Gammaproteobacteria Our findings provide new insights into the microbial catabolism of environmental toxic pyridine derivatives.IMPORTANCE Picolinic acid is a common metabolite of l-tryptophan and some aromatic compounds and is an important intermediate in organic chemical synthesis. Although the microbial degradation/detoxification of picolinic acid has been studied for over 50 years, the underlying molecular mechanisms are still unknown. Here, we show that the pic gene cluster is responsible for the complete degradation of picolinic acid. The pic gene cluster was found to be widespread in other Alpha-, Beta-, and Gammaproteobacteria These findings provide a new perspective for understanding the catabolic mechanisms of picolinic acid in bacteria.

Housefly larvae (Musca domestica) significantly accelerates degradation of monensin by altering the structure and abundance of the associated bacterial community.

Vermicomposting of livestock manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. Monensin (MON), an ionophore polyether antibiotic (IPA), is widely used in broiler feed to control coccidiosis. However, MON residues in litter have become a major source of pollution in the environment. In this work, we studied the efficiency of housefly larvae (Musca domestica) on monensin attenuation during a 12-day laboratory scale vermicomposting experiment. We observed a 94.99% reduction in MON concentration after four days in treatment groups, while it took twelve days to remove more than 94.71% of MON in the control group. We found that the bacterial community composition of the substrate was reshaped by housefly larvae. From the treatment groups, three MON-degrading bacterial strains were isolated and identified as Acinetobacter sp., Stenotrophomonas sp. and Alcaligenes sp. based on 16 S rRNA gene sequence analysis. These three strains were among dominant the bacteria in treated substrates, showing between 52.80% and 89.25% degradation of MON in mineral salt medium within 28 days. Furthermore, two MON-degrading bacteria (Stenotrophomonas sp. and Alcaligenes sp.) were more abundant in treatment groups and larvae gut groups compared with those in control groups. The abundance enhancement of MON-degrading bacteria was related to the change in ambient temperature and pH in the substrates, which were affected by housefly larvae activities. Our results confirm that housefly larvae can significantly accelerate degradation of MON in chicken manure by increasing the abundance of MON-degrading bacteria.

A Novel Degradation Mechanism for Pyridine Derivatives in Alcaligenes faecalis JQ135.

5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of 5HPA metabolism remain unknown. In this study, an operon (hpa), responsible for 5HPA degradation, was cloned from Alcaligenes faecalis JQ135. HpaM was a monocomponent flavin adenine dinucleotide (FAD)-dependent monooxygenase and shared low identity (only 28 to 31%) with reported monooxygenases. HpaM catalyzed the ortho decarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH dependent. The apparent Km values of HpaM for 5HPA and NADH were 45.4 µM and 37.8 µM, respectively. The genes hpaX, hpaD, and hpaF were found to encode 2,5DHP dioxygenase, N-formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation in A. faecalis JQ135. Furthermore, the gene maiA, which encodes a maleic acid cis-trans isomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135, indicating that it might be a key gene in the metabolism of pyridine derivatives. The genes and proteins identified in this study showed a novel degradation mechanism of pyridine derivatives.IMPORTANCE Unlike the benzene ring, the uneven distribution of the electron density of the pyridine ring influences the positional reactivity and interaction with enzymes; e.g., the ortho and para oxidations are more difficult than the meta oxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, the ortho hydroxylations of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while the meta hydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed the ortho decarboxylative hydroxylation of 5HPA. In addition, we found that the maiA gene coding for maleic acid cis-trans isomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives.

Potential for aerobic NO2- reduction and corresponding key enzyme genes involved in Alcaligenes faecalis strain NR.

The potential for aerobic NO2- removal by Alcaligenes faecalis strain NR was investigated. 35 mg/L of NO2--N was removed by strain NR under aerobic conditions in the presence of NH4+. 15N-labeling experiment demonstrated that N2O and N2 were possible products during the aerobic nitrite removal process by strain NR. The key enzyme genes of nirK, norB and nosZ, which regulate the aerobic nitrite denitrification process, were successfully amplified from strain NR. The gene sequence analysis indicates that copper-containing nitrite reductase (NIRK) and periplasmic nitrous oxide reductase (NOSZ) were both hydrophilic protein and the transmembrane structures were absent, while nitric oxide reductase large subunit (NORB) was a hydrophobic and transmembrane protein. According to the three-dimensional structure and binding site analysis, the bulky and hydrophobic methionine residue proximity to the nitrite binding sites of NIRK was speculated to be related to the oxygen tolerance of NIRK from strain NR.

Biodegradation of anionic surfactants by Alcaligenes faecalis, Enterobacter cloacae and Serratia marcescens strains isolated from industrial wastewater.

Pseudo-persistent organic pollutants, such as anionic surfactants (AS), are nowadays among the more complex problems that threaten the aquatic environments and other environmental compartments. The present work describes the identification and efficiency of a consortium, isolated from Algerian industrial wastewater, to remove three anionic surfactants (i.e., sodium dodecylbenzenesulfonate (SDBS), sodium dodecyl sulfate (SDS) and sodium lauryl ether sulfate (SLES)). The genetic analysis of 16S rRNA indicated that these strains are Alcaligenes faecalis, Enterobacter cloacae and Serratia marcescens. Under aerobic conditions, pH 7.0 and optimum temperature of 30 °C, the mixed consortium allowed to degrade 85.1% of initial SDBS amount after 144 h of incubation with half-life of 20.8 h. While E. cloacae and S. marcescens pure strains eliminated 46% and 41% less SDBS respectively. Evenly, SDS was degraded at only 23.71% by A. faecalis strain. However, the degradation capacity of SDS by the consortium was very high (94.2%) with a half-life of 9.8 h. The SLES anionic surfactant showed a lower biodegradation by the consortium (47.53%) due to the presence of ether oxide units in the chemical structure of SLES which induced toxicity to the medium. The investigation of the biodegradation of this type of organic pollutants by microorganisms has recently become a key issue for the environmental protection area.

Selenite Reduction and the Biogenesis of Selenium Nanoparticles by Alcaligenesfaecalis Se03 Isolated from the Gut of Monochamus alternatus (Coleoptera: Cerambycidae).

In this study, a bacterial strain exhibiting high selenite (Na2SeO3) tolerance and reduction capacity was isolated from the gut of Monochamus alternatus larvae and identified as Alcaligenes faecalis Se03. The isolate exhibited extreme tolerance to selenite (up to 120 mM) when grown aerobically. In the liquid culture medium, it was capable of reducing nearly 100% of 1.0 and 5.0 mM Na2SeO3 within 24 and 42 h, respectively, leading to the formation of selenium nanoparticles (SeNPs). Electron microscopy and energy dispersive X-ray analysis demonstrated that A. faecalis Se03 produced spherical electron-dense SeNPs with an average hydrodynamic diameter of 273.8 ± 16.9 nm, localized mainly in the extracellular space. In vitro selenite reduction activity and real-time PCR indicated that proteins such as sulfite reductase and thioredoxin reductase present in the cytoplasm were likely to be involved in selenite reduction and the SeNPs synthesis process in the presence of NADPH or NADH as electron donors. Finally, using Fourier-transform infrared spectrometry, protein and lipid residues were detected on the surface of the biogenic SeNPs. Based on these observations, A. faecalis Se03 has the potential to be an eco-friendly candidate for the bioremediation of selenium-contaminated soil/water and a bacterial catalyst for the biogenesis of SeNPs.

Biodegradation of ochratoxin A by Alcaligenes faecalis isolated from soil.

AIMS: The aim of this study is to report on the hydrolytic action of Alcaligenes faecalis isolated from soil samples and its ability to degrade ochratoxin A. METHODS AND RESULTS: An A. faecalis strain was identified and characterized by employing both a phenotypic analysis and 16S rDNA sequence analysis. The results show that this strain could degrade ochratoxin A efficiently but could not use it as a sole carbon source. Ochratoxin α was confirmed as a degradation product in the intracellular extract of A. faecalis using UPLC-MS/MS. Our results suggest that the biodegradation of ochratoxin A by the A. faecalis strain occurs through the hydrolysis of the ochratoxin A amide bond by a putative peptidase. This is the first report to date on the degradation of ochratoxin A by A. faecalis. CONCLUSION: The A. faecalis strain is presumably a suitable candidate for use in the biodegradation of ochratoxin A. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochratoxin A, which is produced by some filamentous fungi, severely impacts human and animal health by contaminating several types of food and feed. Our study contributes to the identification of the function of A. faecalis 0D-1, which is capable of producing hydrolytic enzyme(s) to biodegrade ochratoxin A into nontoxic ochratoxin α, to minimize the risk associated with ochratoxin A exposure.

Biodegradation of Picolinic Acid by a Newly Isolated Bacterium Alcaligenes faecalis Strain JQ135.

We isolated a bacterial strain JQ135 from municipal wastewater, which was capable of efficiently degrading picolinic acid (PA). Based on the physico-biochemical characteristics and 16S rDNA analysis, strain JQ135 was identified as Alcaligenes faecalis. In addition, strain JQ135 produced an orange pigment when cultured in the Luria-Bertani medium, which is different from the previously reported strains of A. faecalis. During the degradation of PA by the resting strain JQ135 cells, only one intermediate, 6-hydroxypicolinic acid (6HPA), was detected by ultraviolet spectrophotometry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry. A random transposon mutagenesis library of strain JQ135 was constructed. One mutant, Mut-G31, could convert PA into 6HPA without further degradation. The disrupted gene (orf2) was amplified from Mut-G31, and its product showed 32% identity to the 3-deoxy-D-manno-octulosonic acid kinase (KdkA) from Haemophilus influenzae. Results from complementation analysis confirmed that GTG was the initiation codon of the kdkA-like orf2, and that it was essential for PA biodegradation by strain JQ135. This study provides the first genetic evidence for the bacterial degradation of PA.

Metal resistance mechanisms in Gram-negative bacteria and their potential to remove Hg in the presence of other metals.

Contamination of the environment by heavy metals has been increasing in recent years due to industrial activities. Thus research involving microorganisms capable of surviving in multi-contaminated environments is extremely important. The objectives of the present study were to evaluate the removal of mercury alone and in the presence of cadmium, nickel and lead by four mercury-resistant microorganisms; estimate the removal of Cd, Ni and Pb; understand the mechanisms involved (reduction, siderophores, biofilms, biosorption and bioaccumulation) in the metal resistance of the isolate Pseudomonas sp. B50D; and determine the capacity of Pseudomonas sp. B50D in removing Hg, Cd, Ni and Pb from an industrial effluent. It was shown that the four isolates evaluated were capable of removing from 62% to 95% of mercury from a culture medium with no addition of other metals. The isolate Pseudomonas sp. B50D showed the best performance in the removal of mercury when evaluated concomitantly with other metals. This isolate was capable of removing 75% of Hg in the presence of Cd and 91% in the presence of Ni and Pb. With respect to the other metals it removed 60%, 15% and 85% of Cd, Ni and Pb, respectively. In tests with effluent, the isolate Pseudomonas sp. B50D removed 85% of Hg but did not remove the other metals. This isolate presented reduction, biosorption, biofilm production and siderophore production as its metal resistance mechanisms. Pseudomonas sp. B50D was thus a candidate with potential for application in the bioremediation of effluents with complex metal contaminations.

Metal induced changes in trivalent chromium resistant Alcaligenes faecalis VITSIM2.

The changes induced in bacterial strains under stress conditions provide an insight into metal resistance strategies. Trivalent chromium resistant bacterium were isolated and identified by 16S rRNA gene sequencing and designated as Alcaligenes faecalis VITSIM2. The growth pattern was monitored. The organism also showed resistance to copper, cadmium, and certain antibiotics. The differentially expressed proteins in SDS PAGE were identified by mass spectrometry as flagellin and 50S ribosomal L36 protein. The morphological changes were identified by scanning electron microscopy. The changes in the cell wall content were estimated by peptidoglycan analysis and transformation of phosphates was detected by 31 P NMR. Flow cytometry was employed to measure the membrane integrity, esterase activity and intracellular pH. In conclusion spectrum of proteomic, physiological, and morphological alterations was observed that aid the organism to overcome chromium stress.

Enhanced Alcaligenes faecalis Denitrification Rate with Electrodes as the Electron Donor.

The utilization by Alcaligenes faecalis of electrodes as the electron donor for denitrification was investigated in this study. The denitrification rate of A. faecalis with a poised potential was greatly enhanced compared with that of the controls without poised potentials. For nitrate reduction, although A. faecalis could not reduce nitrate, at three poised potentials of +0.06, -0.06, and -0.15 V (versus normal hydrogen electrode [NHE]), the nitrate was partially reduced with -0.15- and -0.06-V potentials at rates of 17.3 and 28.5 mg/liter/day, respectively. The percentages of reduction for -0.15 and -0.06 V were 52.4 and 30.4%, respectively. Meanwhile, for nitrite reduction, the poised potentials greatly enhanced the nitrite reduction. The nitrite reduction rates for three poised potentials (-0.06, -0.15, and -0.30 V) were 1.98, 4.37, and 3.91 mg/liter/h, respectively. When the potentials were cut off, the nitrite reduction rate was maintained for 1.5 h (from 2.3 to 2.25 mg/liter/h) and then greatly decreased, and the reduction rate (0.38 mg/liter/h) was about 1/6 compared with the rate (2.3 mg/liter/h) when potential was on. Then the potentials resumed, but the reduction rate did not resume and was only 2 times higher than the rate when the potential was off.