Enhancement of benzo[a]pyrene mineralization: symbiotic biodegradation by Acinetobacter sp. strain HAP1 in Association with Cyanobacteriota sp. S66.

The ability of Acinetobacter sp. strain HAP1, isolated from petroleum refinery effluent, to eliminate different concentrations (20, 40, 60, 80 and 100 mg/L) of Benzo[a]Pyrene degradation (BaP) was studied. A test to improve this degradation capacity was carried out by culturing the bacterial strain in association with a cyanobacteria. The results show a highly significant effect of the concentration of (BaP) and a very highly significant effect of the symbiosis between the bacterial strain and the cyanobacteria. This combination was able to significantly improve the (BaP) degradation rate by up to 18%. This degradation and especially in association leads to a complete mineralization of (BaP) and there is a difference in yield that can go up to 15%. Through molecular identification based on 16S rRNA gene sequence analysis, strains HAP1 and S66 were recognized as Acinetobacter sp. strain HAP1 and Cyanobacteriota sp. S66, respectively. Comparison of the retrieved sequences with the NCBI GenBank database was done, and the closest matches were found to be Acinetobacter pittii strain JD-10 for bacteria and Pseudochroococcus couteii strain PMC 885.14 for cyanobacteria.

Structural basis of Acinetobacter type IV pili targeting by an RNA virus.

Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics.

Bisphenols can promote antibiotic resistance by inducing metabolic adaptations and natural transformation.

Whether bisphenols, as plasticizers, can influence bacterial uptake of antibiotic resistance genes (ARGs) in natural environment, as well as the underlying mechanism remains largely unknown. Our results showed that four commonly used bisphenols (bisphenol A, S, F, and AF) at their environmental relative concentrations can significantly promote transmission of ARGs by 2.97-3.56 times in Acinetobacter baylyi ADP1. Intriguingly, we observed ADP1 acquired resistance by integrating plasmids uptake and cellular metabolic adaptations other than through reactive oxygen species mediated pathway. Metabolic adaptations including upregulation of capsules polysaccharide biosynthesis and intracellularly metabolic enzymes, which enabled formation of thicker capsules for capturing free plasmids, and degradation of accumulated compounds. Simultaneously, genes encoding DNA uptake and translocation machinery were incorporated to enhance natural transformation of antibiotic resistance carrying plasmids. We further exposed aquatic fish to bisphenols for 120 days to monitor their long-term effects in aquatic environment, which showed that intestinal bacteria communities were dominated by a drug resistant microbiome. Our study provides new insight into the mechanism of enhanced natural transformation of ARGs by bisphenols, and highlights the investigations for unexpectedly-elevated antibiotic-resistant risks by structurally related environmental chemicals.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Changes of MRGs and ARGs in Acinetobacter sp. SL-1 used for treatment of Cr(VI)-contaminated wastewater with waste molasses as carbon source.

Antibiotic resistance genes (ARGs) may be synergistic selected during bio-treatment of chromium-containing wastewater and causing environmental risks through horizontal transfer. This research explored the impact of self-screening bacterium Acinetobacter sp. SL-1 on the treatment of chromium-containing wastewater under varying environmental conditions. The findings indicated that the optimal Cr(VI) removal conditions were an anaerobic environment, 30 °C temperature, 5 g/L waste molasses, 100 mg/L Cr(VI), pH = 7, and a reaction time of 168 h. Under these conditions, the removal of Cr(VI) reached 99.10 %, however, it also developed cross-resistance to tetracycline, gentamicin, clarithromycin, ofloxacin following exposure to Cr(VI). When decrease Cr(VI) concentration to 50 mg/L at pH of 9 with waste molasses as carbon source, the expression of ARGs was down regulated, which decreased the horizontal transfer possibility of ARGs and minimized the potential environmental pollution risk caused by ARGs. The study ultimately emphasized that the treatment of chromium-containing wastewater with waste molasses in conjunction with SL-1 not only effectively eliminates hexavalent chromium but also mitigates the risk of environmental pollution.

Regulation of tricarboxylate transport and metabolism in Acinetobacter baylyi ADP1.

Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa.IMPORTANCEIngestion of Taa and Tcb by grazing livestock can cause a serious metabolic disorder called grass tetany. The disorder, which results from Tcb absorption by ruminants, focuses attention on the metabolism of tricarboxylic acids. Additional interest stems from efforts to produce tricarboxylic acids as commodity chemicals. Improved understanding of bacterial enzymes and pathways for tricarboxylic acid metabolism may contribute to new biomanufacturing strategies.

Obtainment and Inoculation of Acinetobacter pittii Strain JJ-2, and Combined Action with Plants for Formaldehyde and CO2 Removal: A Research Study.

A formaldehyde-degrading bacterium JJ-2 was isolated from the rhizosphere of Chlorophytum and identified as Acinetobacter pittii by colony morphology and 16S rDNA sequence analysis. Further studies showed that under optimal conditions, JJ-2 could maintain activity for six cycles at an initial formaldehyde concentration of 450 mg L-1. At the same time, the complete degradation time was shortened from 12 to 6 h. When the JJ-2 strain was inoculated into sterile soil, the surface spray method had the best effect, and the removal efficiency of 5 ppm formaldehyde increased by 22.63%. In an actual potted plants system colonized with strain JJ-2, the first and second fumigations (without re-inoculation) increased removal by 1.36 times and 0.92 times during the day and 1.27 times and 2.07 times at night. In addition, in the second fumigation, the plant-bacteria combined system was 693.63 ppm and the plant system was 715.34 ppm, effectively reducing the CO2 concentration. This study provides an economical, ecological, and efficient approach to improve the combined system of plants and bacteria to remove gaseous formaldehyde from indoor air, with a positive impact on carbon neutrality.

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification bacterium Acinetobacter ZQ-A1 and community characteristics analysis of its application in pig farm wastewater.

A heterotrophic nitrifying aerobic denitrifying (HN-AD) strain ZQ-A1 with excellent denitrification performance, identified as Acinetobacter, was isolated from simultaneous nitrification and denitrification (SND) craft. ZQ-A1 was capable of removing NH4+, NO2-, and NO3-; the 21-hour removal rates were 84.84%, 87.13%, and 92.63%. ZQ-A1 has the ability to treat mixed nitrogen sources. In addition, ZQ-A1 can be well applied to actual sewage. According to the analysis of microbial community characteristics, the relative abundance of Acinetobacter in the experimental group increased from 0.06% to 2.38%, which is an important reason for the removal rate of NH4+ exceeding 99% within 30 days. The results of KEGG function prediction showed that with the addition of ZQ-A1, the relative abundance of pathways related to bacterial metabolism, such as tricarboxylic acid cycle metabolism, was higher. The research expanded the thinking of HN-AD bacteria in actual production and laid a foundation for its application in sewage treatment.

Induced Expression of the Acinetobacter sp. Oxa Gene in Lactobacillus acidophilus and Its Increased ZEN Degradation Stability by Immobilization.

Zearalenone (ZEN, ZEA) contamination in various foods and feeds is a significant global problem. Similar to deoxynivalenol (DON) and other mycotoxins, ZEN in feed mainly enters the body of animals through absorption in the small intestine, resulting in estrogen-like toxicity. In this study, the gene encoding Oxa, a ZEN-degrading enzyme isolated from Acinetobacter SM04, was cloned into Lactobacillus acidophilus ATCC4356, a parthenogenic anaerobic gut probiotic, and the 38 kDa sized Oxa protein was expressed to detoxify ZEN intestinally. The transformed strain L. acidophilus pMG-Oxa acquired the capacity to degrade ZEN, with a degradation rate of 42.95% at 12 h (initial amount: 20 µg/mL). The probiotic properties of L. acidophilus pMG-Oxa (e.g., acid tolerance, bile salt tolerance, and adhesion properties) were not affected by the insertion and intracellular expression of Oxa. Considering the low amount of Oxa expressed by L. acidophilus pMG-Oxa and the damage to enzyme activity by digestive juices, Oxa was immobilized with 3.5% sodium alginate, 3.0% chitosan, and 0.2 M CaCl2 to improve the ZEN degradation efficiency (from 42.95% to 48.65%) and protect it from digestive juices. The activity of immobilized Oxa was 32-41% higher than that of the free crude enzyme at different temperatures (20-80 °C), pH values (2.0-12.0), storage conditions (4 °C and 25 °C), and gastrointestinal simulated digestion conditions. Accordingly, immobilized Oxa could be resistant to adverse environmental conditions. Owing to the colonization, efficient degradation performance, and probiotic functionality of L. acidophilus, it is an ideal host for detoxifying residual ZEN in vivo, demonstrating great potential for application in the feed industry.

Functional response of Acinetobacter guillouiae SFC 500-1A to tannery wastewater as revealed by a complementary proteomic approach.

Acinetobacter guillouiae SFC 500-1 A is a promising candidate for the bioremediation of tannery wastewater. In this study, we applied shotgun proteomic technology in conjunction with a gel-based assay (Gel-LC) to explore the strain's intracellular protein profile when grown in tannery wastewater as opposed to normal culture conditions. A total of 1775 proteins were identified, 52 of which were unique to the tannery wastewater treatment. Many of them were connected to the degradation of aromatic compounds and siderophore biosynthesis. On the other hand, 1598 proteins overlapped both conditions but were differentially expressed in each. Those that were upregulated in wastewater (109) were involved in the processes mentioned above, as well as in oxidative stress mitigation and intracellular redox state regulation. Particularly interesting were the downregulated proteins under the same treatment (318), which were diverse but mainly linked to the regulation of basic cellular functions (replication, transcription, translation, cell cycle, and wall biogenesis); metabolism (amino acids, lipids, sulphate, energetic processes); and other more complex responses (cell motility, exopolysaccharide production, biofilm formation, and quorum sensing). The findings suggest that SFC 500-1 A engages in survival and stress management strategies to cope with the toxic effects of tannery wastewater, and that such strategies may be mostly oriented at keeping metabolic processes to a minimum. Altogether, the results might be useful in the near future to improve the strain's effectiveness if it will be applied for bioremediation.

Characterization and genome analysis of Acinetobacter oleivorans S4 as an efficient hydrocarbon-degrading and plant-growth-promoting rhizobacterium.

Plant-growth-promoting rhizobacteria (PGPR) have received increasing attention for assisting phytoremediation. However, the effect of PGPR on total petroleum hydrocarbon (TPH) degradation and plant growth promotion and its underlying mechanism is not well understood. In this study, phenotypic analysis and whole genome sequencing were conducted to comprehensively characterize a newly isolated rhizobacterium strain S4, which was identified as Acinetobacter oleivorans, from a TPH-contaminated soil. The strain degraded 62.5% of initially spiked diesel (1%) in minimal media within six days and utilized n-alkanes with a wide range of chain length (i.e., C12 to C40). In addition, the strain showed phenotypic traits beneficial to plant growth, including siderophore production, indole-3-acetic acid synthesis and phosphate solubilization. Potential metabolic pathways and genes encoding proteins responsible for the phenotypic traits were identified. In a real TPH-contaminated soil, inoculation of Acinetobacter oleivorans S4 significantly enhanced the growth of tall fescue relative to the soil without inoculation. In contrast, inoculation of Bacillus sp. Z7, a hydrocarbon-degrading strain, showed a negligible effect on the growth of tall fescue. The removal efficiency of TPH with inoculation of Acinetobacter oleivorans S4 was significantly higher than those without inoculation or inoculation of Bacillus sp. Z7. These results suggested that traits of PGPR beneficial to plant growth are critical to assist phytoremediation. Furthermore, heavy metal resistance genes and benzoate and phenol degradation genes were found in the genome of Acinetobacter oleivorans S4, suggesting its application potential in broad scenarios.

Exploration of the mechanism of 2-CP degradation by Acinetobacter sp. stimulated by Lactobacillus plantarum fermentation waste: A bio-waste reuse.

Green and economical pollution management methods which reusing bio-waste as biostimulant to effectively improve the removal of target pollutants are receiving more and more attention. In this study, Lactobacillus plantarum fermentation waste solution (LPS) was used to investigate its facilitative effect and the stimulation mechanisms on the degradation of 2-chlorophenol (2-CP) by strain Acinetobacter sp. strain ZY1 in terms of both cell physiology and transcriptomics. The degradation efficiency of 2-CP was improved from 60% to > 80% under LPS treatment. The biostimulant maintained the morphology of strain, reduced the level of reactive oxygen species, and recovered the cell membrane permeability from 39% to 22%. It also significantly increased the level of electron transfer activity and extracellular polymeric substances secretion and improved the metabolic activity of the strain. The transcriptome results revealed the stimulation of LPS to promote biological processes such as bacterial proliferation, metabolism, membrane structure composition, and energy conversion. This study provided new insights and references for the reuse of fermentation waste streams in biostimulation methods.

Detoxification of fluoroglucocorticoid by Acinetobacter pittii C3 via a novel defluorination pathway with hydrolysis, oxidation and reduction: Performance, genomic characteristics, and mechanism.

Biological dehalogenation degradation was an important detoxification method for the ecotoxicity and teratogenic toxicity of fluorocorticosteroids (FGCs). The functional strain Acinetobacter pittii C3 can effectively biodegrade and defluorinate to 1 mg/L Triamcinolone acetonide (TA), a representative FGCs, with 86 % and 79 % removal proportion in 168 h with the biodegradation and detoxification kinetic constant of 0.031/h and 0.016/h. The dehalogenation and degradation ability of strain C3 was related to its dehalogenation genomic characteristics, which manifested in the functional gene expression of dehalogenation, degradation, and toxicity tolerance. Three detoxification mechanisms were positively correlated with defluorination pathways through hydrolysis, oxidation, and reduction, which were regulated by the expression of the haloacid dehalogenase (HAD) gene (mupP, yrfG, and gph), oxygenase gene (dmpA and catA), and reductase gene (nrdAB and TgnAB). Hydrolysis defluorination was the most critical way for TA detoxification metabolism, which could rapidly generate low-toxicity metabolites and reduce toxic bioaccumulation due to hydrolytic dehalogenase-induced defluorination. The mechanism of hydrolytic defluorination was that the active pocket of hydrolytic dehalogenase was matched well with the spatial structure of TA under the adjustment of the hydrogen bond, and thus induced molecular recognition to promote the catalytic hydrolytic degradation of various amino acid residues. This work provided an effective bioremediation method and mechanism for improving defluorination and detoxification performance.

Genomics and nitrogen metabolic characteristics of a novel heterotrophic nitrifying-aerobic denitrifying bacterium Acinetobacter oleivorans AHP123.

A novel aerobic strain of Acinetobacter oleivorans AHP123 was isolated from activated sludge, which could conduct heterotrophic nitrification and denitrification simultaneously. This strain has excellent NH4+-N removal ability, with 97.93% removal rate at 24-hour. To identify the metabolic pathways of this novel strain, genes of gam, glnA, gdhA, gltB, nirB, nasA, nar, nor, glnK and amt were detected by genome analysis. Through RT-qPCR, it was found that the expression of key genes confirmed two possible ways of nitrogen removal in strain AHP123: nitrogen assimilation and heterotrophic nitrification aerobic denitrification (HNAD). However, the absence of some common HNAD genes (amo, nap and nos) suggested that strain AHP123 might have a different HNAD pathway from other HNAD bacteria. Nitrogen balance analysis revealed that strain AHP123 assimilated most of the external nitrogen sources into intracellular nitrogen.

Overproduction, purification, and transcriptional activity of recombinant Acinetobacter baylyi ADP1 RNA polymerase holoenzyme.

Acinetobacter baylyi is an interesting model organism to investigate bacterial metabolism due to its vast repertoire of metabolic enzymes and ease of genetic manipulation. However, the study of gene expression in vitro is dependent on the availability of its RNA polymerase (RNAp), an essential enzyme in transcription. In this work, we developed a convenient method of producing the recombinant A. baylyi ADP1 RNA polymerase holoenzyme (RNApholo) in E. coli that yields 22 mg of a >96% purity protein from a 1-liter shake flask culture. We further characterized the A. baylyi ADP1 RNApholo kinetic profile using T7 Phage DNA as template and demonstrated that it is a highly transcriptionally active enzyme with an elongation rate of 24 nt/s and a termination efficiency of 94%. Moreover, the A. baylyi ADP1 RNApholo has a substantial sequence identity (∼95%) with the RNApholo from the human pathogen Acinetobacter baumannii. This protein can serve as a source of material for structural and biological studies towards advancing our understanding of genome expression and regulation in Acinetobacter species.

Growth phase-dependent production of the adhesive nanofiber protein AtaA in Acinetobacter sp. Tol 5.

AtaA, the sticky, long, and peritrichate nanofiber protein from Acinetobacter sp. Tol 5, mediates autoagglutination and is highly adhesive to various material surfaces, resulting in a biofilm. Although the production of the adhesive nanofiber protein is likely to require a large amount of energy and material sources, the relationship between AtaA fiber production and cell growth remains unknown. Here, we report the growth phase-dependent AtaA fiber production in Tol 5. We examined the ataA gene expression in different growth phases using a reporter gene assay with an originally developed reporter plasmid and using reverse transcription-quantitative polymerase chain reaction. Bacterial cells with surface-displayed AtaA at different growth phases were immunostained and analyzed using fluorescence flow cytometry and confocal laser scanning microscopy. The results indicate that Tol 5 modulated the amount of surface-displayed AtaA at the transcriptional level. AtaA production was low in the early growth phase but remarkably increased in the late growth phase, covering the whole bacterial cell with AtaA fibers in the stationary phase. Tol 5 displayed AtaA fibers poorly in the early growth phase and showed less autoagglutination and adhesiveness than those in the stationary phase. Although Tol 5 grew as fast as its ataA-deficient mutant in the early growth phase, the optical density of Tol 5 culture was slightly lower than that of the ataA-deficient mutant in the late growth phase. Based on these experimental results, we propose the growth-phase-dependent production of AtaA fiber for efficient and fast cell growth.

Characterization of new diesel-degrading bacteria isolated from freshwater sediments.

As the result of diesel's extensive production and use as fuel for transportation, pollution with such complex mixtures of hydrocarbons is a major concern worldwide. The present study's focus was to investigate the presence of diesel-degrading bacteria in different Danube Delta freshwater sediments. Ten bacterial strains capable to grow in a minimal medium with diesel as the sole carbon source were isolated and characterized in this study. Based on the phenotypic and molecular characteristics, the ten strains belong to four genera and seven species, such as Pseudomonas (P. aeruginosa, P. nitroreducens, P. resinovorans, P. multiresinivorans), Acinetobacter (A. tandoii), Bacillus (B. marisflavi), and Stenotrophomonas (S. maltophilia). All these bacteria were excellent biosurfactant producers, and they were able to tolerate saturated hydrocarbons, like n-heptane, n-decane, n-pentadecane, and n-hexadecane. The ten strains possess at least one alkane hydroxylase gene in their genome, and they were also able to tolerate and degrade diesel. Higher biodegradation rates of diesel were acquired for the strains from the genera Pseudomonas, Acinetobacter, and Stenotrophomonas, compared with that obtained for the Bacillus strain. Due to their remarkable potential to degrade diesel and produce biosurfactants, the ten isolated bacteria are attractive candidates for bioremediation of diesel-polluted environments.

Organic and inorganic nitrogen removals by an ureolytic heterotrophic nitrification and aerobic denitrification strain Acinetobacter sp. Z1: Elucidating its physiological characteristics and metabolic mechanisms.

Although heterotrophic nitrification-aerobic denitrification (HN-AD) is promising in nitrogen removal, it remains unclear for most HN-AD strains in physiological characteristics and metabolic mechanisms. In this study, a newly isolated strain Acinetobacter sp. Z1 converted not only inorganic nitrogen, but also organic nitrogen to N2. Among them, urea was the preferential nitrogen substrate. Single-factor experiments showed that efficient HN-AD process occurred with acetate as carbon source, C/N ratios of 12 for NH4+-N and 15 for NO3--N, pH 8, 30 °C, DO of ∼5.8 mg/L and salinity less than 1.5 %. Subsequently, response surface analysis was applied to predict the optimal growth conditions. Its complete genome annotation in combination with enzymatic activity assay and nitrogen balance calculation showed that at least four pathways involved in nitrogen metabolism. This work indicates that ureolytic strain Z1 could be prepared as bacterial agents with other HN-AD strains to treat urea-containing wastewater like urine from urban community.

Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1.

Microbial production of intracellular compounds can be engineered by redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)-based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We first established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Second, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock-outs to generate significantly enlarged filamentous or spherical cells respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids.

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Phylogenetic analysis, homology modelling and biochemical methods have been employed to characterize a phytase from a Gram-negative soil bacterium. Acinetobacter sp. AC1-2 phytase belongs to clade 2 of the histidine (acid) phytases, to the Multiple Inositol Polyphosphate Phosphatase (MINPP) subclass. The enzyme was extraordinarily stable in solution both at room temperature and 4°C, retaining near 100% activity over 755 days. It showed a broad pH activity profile from 2-8.5 with maxima at 3, 4.5-5 and 6. The enzyme showed Michaelis-Menten kinetics and substrate inhibition (Vmax, Km, and Ki, 228 U/mg, 0.65 mM and 2.23 mM, respectively). Homology modelling using the crystal structure of a homologous MINPP from a human gut commensal bacterium indicated the presence of a potentially stabilising polypeptide loop (a U-loop) straddling the active site. By employ of the enantiospecificity of Arabidopsis inositol tris/tetrakisphosphate kinase 1 for inositol pentakisphosphates, we show AC1-2 MINPP to possess D6-phytase activity, which allowed modelling of active site specificity pockets for InsP6 substrate. While phytase gene transcription was unaltered in rich media, it was repressed in minimal media with phytic acid and orthophosphate as phosphate sources. The results of this study reveal AC1-2 MINPP to possess desirable attributes relevant to biotechnological use.