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A straightforward synthesis of the pentasaccharide with a readily available linker arm corresponding to the O-antigenic polysaccharide of Acinetobacter junii strain 65 has been achieved in good yield. The synthesis has been carried out using thioglycosides as glycosyl donor in the presence of a combination of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as thiophilic activator. The yields of the glycosylation steps were very good with satisfactory stereochemistry at the glycosidic linkages. The pentasaccharide derivative has also been obtained using a one-pot iterative glycosylation strategy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet 1 . Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics 2-6 . Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Biogenic nanoparticles are gaining attention due to their low toxicity and numerous biomedical applications. Present study aimed to compare the potential anticancer activity of two biogenic silver nanoparticles (bAgNPs and pAgNPs) against human cervical cancer cell lines (HeLa)., Methods: bAgNPs were synthesized using Acinetobacter sp. whereas pAgNPs were synthesized using aqueous root extract of Curcuma aromatica. Effect of these nanoparticles on HeLa cells viability was studied using MTT assay and colony formation assay. Anticancer potential was determined using fluorescence microscopy and flow cytometry studies. Bio-compatibility studies were performed against peripheral blood mononuclear cells (PBMCs)., Results: Both the nanoparticles showed 50 % viability of peripheral blood mononuclear cells (PBMCs) when used at high concentration (200 μg/mL). IC 50 for bAgNPs and pAgNPs against HeLa cells were 17.4 and 14 μg/mL respectively. Colony formation ability of Hela cells was reduced on treatment with both nanoparticles. Acridine orange and ethidium bromide staining demonstrated that bAgNPs were cytostatic whereas pAgNPs were apoptotic. JC-1 dye staining revealed that the mitochondrial membrane potential was affected on treatment with pAgNPs while it remained unchanged on bAgNPs treatment. Flow cytometry confirmed cell cycle arrest in HeLa cells on treatment with nanoparticles further leading to apoptosis in case of pAgNPs. About 77 and 58 % HeLa cells were found in subG1 phase on treatment with bAgNPs and pAgNPs respectively. bAgNPs showed cytostatic effect on HeLa cells arresting the cell growth in subG1 phase, whereas, pAgNPs triggered death of HeLa cells through mitochondrial membrane potential impairment and apoptosis., Conclusion: Overall, bAgNPs and pAgNPs could be safe and showed potential to be used as anticancer nano-antibiotics against human cervical cancer cells., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin (1), butanochelin (2), and pentanochelin (3). This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(iii) and may be further modified via thiol-ene or azide-alkyne click chemistry.

Environmental monitoring and potable water control are key applications where optical fiber sensing solutions can outperform other technologies. In this work, we report a highly sensitive plasmonic fiber-optic probe that has been developed to determine the concentration of cadmium ions (Cd 2+ ) in solution. This original sensor was fabricated by immobilizing the Acinetobacter sp. around gold-coated tilted fiber Bragg gratings (TFBGs). To this aim, the immobilization conditions of bacteria on the gold-coated optical fiber surface were first experimentally determined. Then, the coated sensors were tested in vitro. The relative intensity of the sensor response experienced a change of 1.1 dB for a Cd 2+ concentration increase from 0.1 to 1000 ppb. According to our test procedure, we estimate the experimental limit of detection to be close to 1 ppb. Cadmium ions strongly bind to the sensing surface, so the sensor exhibits a much higher sensitivity to Cd 2+ than to other heavy metal ions such as Pb 2+ , Zn 2+ and CrO 4 2- found in contaminated water, which ensures a good selectivity.

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli . The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

Crude glycerol is an excellent carbon source for bacterial production systems. Bacterial fermentation often generates by-products that can offer an additional carbon pool to improve the product profile for optimal valorization. In this study, the properties of two phylogenetically distinct bacteria, Acinetobacter baylyi ADP1 and Clostridium butyricum, were coupled in a one-pot batch process to co-produce 1,3 propanediol (PDO) and long-chain alkyl esters (wax esters, WEs) from crude glycerol. In the process, A. baylyi deoxidized the growth medium allowing glycerol fermentation and PDO production by C. butyricum. Reaeration of the co-cultivations enabled A. baylyi to metabolize the fermentation by-products, acetate and butyrate, and synthesize intracellular WEs. To improve PDO production and A. baylyi growth, carbon and macronutrients in the growth medium were screened and optimized using Plackett-Burman and Box-Behnken models. The validation experiment revealed a good correlation between the observed and predicted values. The salting-out method recovered 89.5% PDO from the fermentation broth and in vacuo extraction resulted in a PDO content of 5.3 g L -1 . Nuclear magnetic resonance revealed a WE content and yield of 34.4 ± 1.4 mg L -1 and 34.2 ± 3.2 mg WE g -1 dry cell weight, respectively. A molar yield of 0.65 mol PDO mol -1 and 0.62 μmol WE mol -1 crude glycerol was achieved with the synthetic consortium. This work emphasizes the strength of response surface methodology in improving production processes from the mutualistic association of divergent bacterial species in consortium. The co-production of PDO and WEs from crude glycerol is demonstrated for the first time in this study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The potential for coupling bioaugmentation with phytoremediation to simultaneously treat and utilize treated palm oil mill effluent (TPOME) in animal feed production was determined from a reduction in phenolic compounds and color in soil leachates, as well as from an increased yield of pasture grass. Two phenol-degrading bacteria-Methylobacterium sp. NP3 and Acinetobacter sp. PK1-were inoculated into the Brachiaria humidicola rhizosphere before the application of TPOME. A pot study showed that the soil with both grass and inoculated bacteria had the highest dephenolization and decolorization efficiencies, with a maximum capability of removing 70% from 587 mg total phenolic compounds added and 73% from 4438 color units during ten TPOME application cycles. The results corresponded to increases in the number of phenol-degrading bacteria and the grass yield. In a field study, this treatment was able to remove 46% from 21,453 mg total phenolic compounds added, with a maximum color removal efficiency of 52% from 5105 color units, while the uninoculated plots removed about 24-39% and 29-46% of phenolic compounds and color, respectively. The lower treatment performance was probably due to the increased TPOME concentrations. Based on the amounts of phenolic compounds, protein, and crude fiber in the grass biomass, the inoculated TPOME-treated grass had a satisfactory nutritional quality and digestibility for use as animal feed.

Acinetobacter baylyi ADP1 is a microorganism with the potential to produce storage lipids. Here, a systematic study was carried out to evaluate growth performance and accumulation of wax esters and triacylglycerols using glycerol, xylose, glucose, acetate, ethanol, and pyruvate as carbon sources. High specific growth rates (μ) were found in gluconeogenic carbon sources (ethanol, acetate, and pyruvate: 0.94 ± 0.18, 0.93 ± 0.06, and 0.61 ± 0.01 h -1 , respectively), and low in glucose (0.25 ± 0.01 h -1 ). Interestingly, these μ values were sustained in a broad range of concentrations of glucose (0.5-50 g L -1 ), pyruvate (3-10 g L -1 ), and acetate (0.3-2 g L -1 ), suggesting a high tolerance to glucose and pyruvate. It was observed that ADP1 is not able to use glycerol or xylose as unique carbon sources. On the other hand, ADP1 showed sensitivity to osmotic upshifts, noted by the lysis at the beginning of cultivations on different carbon sources. However, ADP1 is adapted to relatively high substrate concentrations as indicated by the minimal inhibitory concentrations (MICs) determined at 24 h of cultivations: 350, 50, 80, and 15 g L -1 for glucose, ethanol, pyruvate, and acetate, respectively. Remarkably, ADP1 co-utilized glucose, acetate, ethanol, and pyruvate. Finally, the accumulation of storage lipids, wax esters (WEs), and triacylglycerols (TAGs) showed to be substrate dependent. Under nitrogen-limiting conditions, the TAGs:WEs (mol:mol) accumulation ratios were 1:4.9 in pyruvate and 1:1.6 in glucose, the WEs were mainly accumulated in acetate. In ethanol, no accumulation of lipids was detected.

We studied the taxonomic position of six phenetically related strains of the genus Acinetobacter, which were recovered from hospital sewage in China and showed different patterns of resistance to clinically important antibiotics. Whole-genome sequencing of these strains and genus-wide phylogeny reconstruction based on a set of 107 Acinetobacter core genes indicated that they formed a separate and internally cohesive clade within the genus. The average nucleotide identity based on BLAST and digital DNA-DNA hybridization values between the six new genomes were 97.25-98.67% and 79.2-89.3%, respectively, whereas those between them and the genomes of the known species were ≤78.57% and ≤28.5%, respectively. The distinctness of the strains at the species level was also supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by MALDI-TOF MS. Moreover, the strains displayed a catabolically unique profile and could be differentiated from the phylogenetically closest species at least by their inability to grow on d,l-lactate. A total of 18 different genes were found in the six genome sequences which encode resistance to seven classes of antimicrobial agents, including clinically important carbapenems, oxyimino-cephalosporins, or aminoglycosides. These genes occurred in five different combinations, with three to 10 different genes per strain. We conclude that the six strains represent a novel Acinetobacter species, for which we propose the name Acinetobacter cumulans sp. nov. to reflect its ability to acquire and cumulate diverse resistance determinants. The type strain is WCHAc060092 T (ANC 5797 T =CCTCC AB 2018119 T =GDMCC 1.1380 T =KCTC 62576 T )., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Introduction: Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism., Objectives: This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1)., Methods: We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D- and 2D-NMR ( 1 H, 13 C, 1 H- 1 H-COSY, 1 H- 13 C-HSQC, 1 H- 13 C-HMBC and 1 H- 15 N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation., Results: We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the millimolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium., Conclusion: Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.

Two new alkaloids indolepyrazines A ( 1 ) and B ( 2 ) were isolated from the marine-derived Acinetobacter sp. ZZ1275. Their structures were elucidated through extensive nuclear magnetic resonance (NMR) spectroscopic analyses, high resolution electrospray ionization mass spectroscopy (HRESIMS) data, and electronic circular dichroism (ECD) calculation. Indolepyrazine A represents the first example of alkaloids with an indole-pyrazine-oxindole skeleton. Both 1 and 2 showed antimicrobial activities against methicillin-resistant Staphylococcus aureus , Escherichia coli , and Candida albicans with minimum inhibitory concentration (MIC) values of 12 μg/mL, 8⁻10 μg/mL, and 12⁻14 μg/mL, respectively.

Background: The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens., Objectives: The present study aimed to verify the occurrence of metallo-β-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region., Methods: Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing., Findings: A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86., Main Conclusions: To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.

Bacterial contact-dependent growth inhibition (CDI) systems are two-partner secretion systems in which toxic CdiA proteins are exported on the outer membrane by cognate transporter CdiB proteins. Upon binding to specific receptors, the C-terminal toxic (CT) domain, detached from CdiA, is delivered to neighbouring cells. Contacts inhibit the growth of not-self-bacteria, lacking immunity proteins co-expressed with CdiA, but promote cooperative behaviours in "self" bacteria, favouring the formation of biofilm structures. The Acinetobacter baylyi ADP1 strain features two CdiA, which differ significantly in size and have different CT domains. Homologous proteins sharing the same CT domains have been identified in A. baumannii. The growth inhibition property of the two A. baylyi CdiA proteins was supported by competition assays between wild-type cells and mutants lacking immunity genes. However, neither protein plays a role in biofilm formation or adherence to epithelial cells, as proved by assays carried out with knockout mutants. Inhibitory and stimulatory properties may be similarly uncoupled in A. baumannii proteins.

Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.

Currently, the capability of identification for Acinetobacter species using MALDI-TOF MS still remains unclear in clinical laboratories due to certain elusory phenomena. Thus, we conducted this research to evaluate this technique and reveal the causes of misidentification. Briefly, a total of 788 Acinetobacter strains were collected and confirmed at the species level by 16S rDNA and rpoB sequencing, and subsequently compared to the identification by MALDI-TOF MS using direct smear and bacterial extraction pretreatments. Cluster analysis was performed based on the mass spectra and 16S rDNA to reflect the diversity among different species. Eventually, 19 Acinetobacter species were confirmed, including 6 species unavailable in Biotyper 3.0 database. Another novel species was observed, temporarily named A. corallinus. The accuracy of identification for Acinetobacter species using MALDI-TOF MS was 97.08% (765/788), regardless of which pretreatment was applied. The misidentification only occurred on 3 A. parvus strains and 20 strains of species unavailable in the database. The proportions of strains with identification score ≥ 2.000 using direct smear and bacterial extraction pretreatments were 86.04% (678/788) and 95.43% (752/788), χ 2  = 41.336, P < 0.001. The species similar in 16 rDNA were discriminative from the mass spectra, such as A. baumannii & A. junii, A. pittii & A. calcoaceticus, and A. nosocomialis & A. seifertii. Therefore, using MALDI-TOF MS to identify Acinetobacter strains isolated from clinical samples was deemed reliable. Misidentification occurred occasionally due to the insufficiency of the database rather than sample extraction failure. We suggest gene sequencing should be performed when the identification score is under 2.000 even when using bacterial extraction pretreatment. Graphical Abstract ᅟ.

In the present study the produced biosurfactant of Acinetobacter junii B6 (recently isolated from Iranian oil excavation site) were partially purified and identified by high performance thin layer chromatography (HPTLC), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance ( 1 H NMR). Elemental analysis of the biosurfactant by energy dispersive X-ray spectroscopy (EDS) revealed that the biosurfactant was anionic in nature. The physiochemical properties of the lipopeptide biosurfactant were evaluated by determination of its critical micelle concentration (CMC) and hydrophile-lipophile balance (HLB). The produced biosurfactant decreased the surface tension of water to 36mNm -1 with the CMC of approximately 300mg/l. Furthermore, the solubility properties of the biosurfactant (dissolved in phosphate-buffer saline solution, pH7.4) were investigated by turbidity examination, dynamic light scattering (DLS) measurements, and transmission electron microscopy (TEM) inspection. It could be concluded that the biosurfactant showed the spherical-shaped vesicles at a concentration higher than its CMC and the circular dichroism (CD) spectra showed that the secondary structure of the biosurfactant vesicles is dominated by the β sheet., (Copyright © 2018. Published by Elsevier B.V.)

Bacteria inhabit a vast range of biological niches and have evolved diverse mechanisms to cope with environmental stressors. The genus Acinetobacter comprises a complex group of Gram-negative bacteria. Some of these bacteria such as A. baumannii are nosocomial pathogens. They are often resistant to multiple antibiotics and are associated with epidemic outbreaks. A. radioresistens is generally considered to be a commensal bacterium on human skin or an opportunistic pathogen. Interestingly, this species has exceptional resistance to a range of environmental challenges which contributes to its persistence in clinical environment and on human skin. We studied changes in its lipid composition induced by the onset of stationary phase. This strain produced triglycerides (TG) as well as four common phospholipids: phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL) and lysocardiolipin (LCL). It also produced small amounts of acyl-phosphatidylglycerol (APG). As the bacterial growth entered the stationary phase, the lipidome switched from one dominated by PE and PG to another dominated by CL and LCL. Surprisingly, bacteria in the stationary phase produced N-acyl-phosphatidylethanolamine (NAPE) and another rare lipid we tentatively name as 1-phosphatidyl-2-acyl-glycero-3-phosphoethanolamine (PAGPE) based on tandem mass spectrometry. It is possible these tri-acylated lipids play an important role in coping with nutrient depletion.

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoprotein Ec LpoB was identified as a critical activator of Escherichia coli PBP1b ( Ec PBP1b), one of the major PG synthases of this organism. Activation of Ec PBP1b is mediated through the association of Ec LpoB with a regulatory domain on Ec PBP1b called UB2H. Notably, Pseudomonas aeruginosa also encodes PBP1b ( Pa PBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potential Pa PBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity of Pa PBP1b. We named this protein LpoP and found that it interacts directly with Pa PBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated that Pa LpoP- Pa PBP1b as well as an equivalent protein pair from Acinetobacter baylyi can fully substitute for Ec LpoB- Ec PBP1b in E. coli for PG synthesis. Furthermore, we show that amino acid changes in Pa PBP1b that bypass the Pa LpoP requirement map to similar locations in the protein as changes promoting Ec LpoB bypass in Ec PBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to synthesize selenium nanoparticles (SeNPs) using cell suspension and total cell protein of Acinetobacter sp. SW30 and optimize its synthesis by studying the influence of physiological and physicochemical parameters. Also, we aimed to compare its anticancer activity with that of chemically synthesized SeNPs in breast cancer cells. Cell suspension of Acinetobacter sp. SW30 was exposed to various physiological and physicochemical conditions in the presence of sodium selenite to study their effects on the synthesis and morphology of SeNPs. Breast cancer cells (4T1, MCF-7) and noncancer cells (NIH/3T3, HEK293) were exposed to different concentrations of SeNPs. The 18 h grown culture with 2.7×10 9 cfu/mL could synthesize amorphous nanospheres of size 78 nm at 1.5 mM and crystalline nanorods at above 2.0 mM Na 2 SeO 3 concentration. Polygonal-shaped SeNPs of average size 79 nm were obtained in the supernatant of 4 mg/mL of total cell protein of Acinetobacter sp. SW30. Chemical SeNPs showed more anticancer activity than SeNPs synthesized by Acinetobacter sp. SW30 (BSeNPs), but they were found to be toxic to noncancer cells also. However, BSeNPs were selective against breast cancer cells than chemical ones. Results suggest that BSeNPs are a good choice of selection as anticancer agents., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials., Results: We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step., Conclusions: We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.

Phenol is a hazardous chemical known to be widely distributed in aquatic environments. Biodegradation is an attractive option for removal of phenol from water sources. Acinetobacter sp. DW-1 isolated from drinking water biofilters can use phenol as a sole carbon and energy source. In this study, we found that Immobilized Acinetobacter sp. DW-1cells were effective in biodegradation of phenol. In addition, we performed proteome and transcriptome analysis of Acinetobacter sp. DW-1 during phenol biodegradation. The results showed that Acinetobacter sp. DW-1 degrades phenol mainly by the ortho pathway because of the induction of phenol hydroxylase, catechol-1,2-dioxygenase. Furthermore, some novel candidate proteins (OsmC-like family protein, MetA-pathway of phenol degradation family protein, fimbrial protein and coenzyme F390 synthetase) and transcriptional regulators (GntR/LuxR/CRP/FNR/TetR/Fis family transcriptional regulator) were successfully identified to be potentially involved in phenol biodegradation. In particular, MetA-pathway of phenol degradation family protein and fimbrial protein showed a strong positive correlation with phenol biodegradation, and Fis family transcriptional regulator is likely to exert its effect as activators of gene expression. This study provides valuable clues for identifying global proteins and genes involved in phenol biodegradation and provides a fundamental platform for further studies to reveal the phenol degradation mechanism of Acinetobacter sp.

Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu&#95;0438, Maqu&#95;3316, Maqu&#95;3410, Maqu&#95;3572, and the enzyme reported under RefSeq accession no. WP&#95;004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu&#95;3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD + cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided. IMPORTANCE This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD + are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn., (Copyright © 2017 American Society for Microbiology.)

Alkanes are one of the most widespread contaminants in the natural environment, primarily as a consequence of biological synthesis and oil spills. Many indigenous microbes metabolize alkanes, and the chemotaxis and accumulation in some strains has been identified. For the first time, we apply Raman microspectroscopy to identify such chemotaxis-related affinity, and quantify the alkane concentrations via spectral alterations. Raman spectral alterations were only found for the alkane chemo-attractant bacteria Acinetobacter baylyi ADP1, not for Pseudomonas fluorescence, which exhibits limited chemotaxis toward alkane. The significant alterations were attributed to the strong chemotactic ability of A. baylyi enhancing the affinity and accumulation of alkane molecules on cell membranes or cellular internalization. Spectral fingerprints of A. baylyi significantly altered after 1-h exposure to pure alkanes (dodecane or tetradecane) and alkane mixtures (mineral oil or crude oil), but not monocyclic aromatic hydrocarbons (MAHs) or polycyclic aromatic hydrocarbons (PAHs). A semilog linear regression relationship between Raman spectral alterations and alkane concentrations showed its feasibility in quantifying alkane concentration in environmental samples. Pure alkanes or alkane mixtures exhibited different limits of detection and regression slopes, indicating that the chemotaxis-related alkane accumulation in A. baylyi is dependent on the carbon chain length. This work provides a novel biospectroscopy approach to characterize the chemotaxis-related alkane bioaccumulation, and has immense potential for fast and high-throughput screening bacterial chemotaxis.

Objectives: Rapid identification of Acinetobacter species is critical as members of the A. baumannii (Ab) group differ in antibiotic susceptibility and clinical outcomes. A. baumannii, A. pittii, and A. nosocomialis can be identified by MALDI-TOF/MS, while the novel species A. seifertii and A. dijkshoorniae cannot. Low identification rates for A. nosocomialis also have been reported. We evaluated the use of MALDI-TOF/MS to identify isolates of A. seifertii and A. dijkshoorniae and revisited the identification of A. nosocomialis to update the Bruker taxonomy database., Methods: Species characterization was performed by rpoB-clustering and MLSA. MALDI-TOF/MS spectra were recovered from formic acid/acetonitrile bacterial extracts overlaid with α-cyano-4-hydroxy-cinnamic acid matrix on a MicroflexLT in linear positive mode and 2000-20 000 m/z range mass. Spectra were examined with the ClinProTools v2.2 software. Mean spectra (MSP) were created with the BioTyper software., Results: Seventy-eight Acinetobacter isolates representative of the Ab group were used to calculate the average spectra/species and generate pattern recognition models. Species-specific peaks were identified for all species, and MSPs derived from three A. seifertii, two A. dijkshoorniae, and two A. nosocomialis strains were added to the Bruker taxonomy database, allowing successful identification of all isolates using spectra from either bacterial extracts or direct colonies, resulting in a positive predictive value (PPV) of 99.6% (777/780) and 96.8% (302/312), respectively., Conclusions: The use of post-processing data software identified statistically significant species-specific peaks to generate reference signatures for rapid accurate identification of species within the Ab group, providing relevant information for the clinical management of Acinetobacter infections., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Two new sesquiterpenes, microsphaeropsisin B ( 1 ) and C ( 2 ), and two new de- O -methyllasiodiplodins, (3 R , 7 R )-7-hydroxy-de- O -methyllasiodiplodin ( 4 ) and (3 R )-5-oxo-de- O -methyllasiodiplodin ( 5 ), together with one new natural product ( 6 ) and twelve known compounds ( 3 , 7 - 17 ), were isolated from the co-cultivation of mangrove endophytic fungus Trichoderma sp. 307 and aquatic pathogenic bacterium Acinetobacter johnsonii B2. Their structures, including absolute configurations, were elucidated by extensive analysis of spectroscopic data, electronic circular dichroism, Mo₂(AcO)₄-induced circular dichroism, and comparison with reported data. All of the isolated compounds were tested for their α-glucosidase inhibitory activity and cytotoxicity. New compounds 4 and 5 exhibited potent α-glucosidase inhibitory activity with IC 50 values of 25.8 and 54.6 µM, respectively, which were more potent than the positive control (acarbose, IC 50 = 703.8 µM). The good results of the tested bioactivity allowed us to explore α-glucosidase inhibitors in lasiodiplodins.

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

A novel, phosphate-binding exobiopolymer (EBP) produced by Acinetobacter haemolyticus MG606 was characterized and its biocompatibility evaluated in RAW 264.7 cells and in mice. EBP was identified as a 50 kDa heteropolysaccharide composed of pentose and hexose sugars. EBP exhibited cytotoxicity, stimulation of free radical production and loss of mitochondrial and lysosomal integrity in RAW 264.7 cells at 500 μg/mL concentration while lower concentrations exhibited no significant (p > 0.05) effect on these parameters. EBP exhibited dose-dependent mortality, body weight reduction, hypothermia and clinical signs of toxicity in mice following intraperitoneal administration. The LD 50 of EBP was determined to be 92.31 mg/kg. Overall, the results of our study suggest that composition of EBP produced by A. haemolyticus MG606 is distinct from EBP produced by other Acinetobacter spp. The high biocompatibility supports application of EBP as a safe biosorbent for phosphate remediation.

This study evaluated five extracellular polymers of bacterial origin possessing mucoadhesive properties for their antimicrobial properties and toxicological characteristics. Of the five tested mucoadhesive biopolymers, the extracellular polymer produced by a strain of Acinetobacter haemolyticus exhibited broad antimicrobial efficacy towards Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7 and Bacillus subtilis. Significant (p<0.05) inhibition of gram negative bacterial pathogens followed by gram positives were observed with the biopolymer at a dose of 40-60μg ml-1 at ambient temperature. The cytotoxicity under in vitro conditions and oral toxicity in murine models was also evaluated. The biopolymer did not elicit either haemolytic activity or toxicity in RAW 264.7 cell lines. Haemotological, histopathological and general examinations indicated no adverse effects in Swiss albino mice fed with the biopolymer (120mg kg-1 body weight-1 day1) over a period of 30 days. These results suggested that the biopolymer was well tolerated without any signs of toxicity and may have several potential biomedical applications where disinfection is desired., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition-including metabolic water produced as a byproduct of metabolism-based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the (18)O/(16)O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications.

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA&#95;C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Two-dimensional gel electrophoresis was conducted to investigate the effect of H2O2 on whole protein expression in Acinetobacter oleivorans DR1. Functional classification of 13 upregulated proteins using MALDI-TOF mass spectrometry showed relationships with oxidative stress, energy production and conversion, nucleotide and amino acid metabolism, membrane-related, ion transport, and chaperone-related functions. Alignment of OxyR-binding regions from Pseudomonas aeruginosa and Escherichia coli with promoters of identified proteins revealed that only ahpC, ahpF, and trxB (thioredoxin-disulfide reductase) genes, along with a newly found oprC (putative outer membrane receptor protein) gene, have OxyR-binding sites. The oxyR and ahpC mutants were more sensitive to H2O2 and showed growth defects in both nutritional and n-hexadecane-amended media. Four catalases present in the genome of A. oleivorans DR1 were not detected, which led us to confirm the expression and activity of those catalases in the presence of H2O2. The expression patterns of the four catalase genes differed at different concentrations of H2O2. Interestingly, the promoters of both known OxyR-controlled katG gene (AOLE&#95;17390) and putative small catalase gene (AOLE&#95;09800) have OxyR-binding sites. Gel-shift assay confirmed OxyR binding to the promoter regions of newly identified OxyR-controlled genes encoding OprC and a putative catalase. Hierarchical expression and OxyR-binding of several OxyR-controlled genes suggested that concentration is an important factor in inducing the set of genes under H2O2 stress.

The present study evaluated an extracellular, novel biopolymer produced by Acinetobacter haemolyticus MG606 for its physicochemical properties and phosphate binding mechanism. The exobiopolymer (EBP) was characterized to be majorly polysaccharide in nature consisting of 48.9 kDa heteropolysaccharide composed of galactose, glucose, xylose, lyxose, allose, ribose, arabinose, mannose and fructose. Maximum phosphate binding efficiency of 25mg phosphate/g of EBP was described by Langmuir isotherm and further, the physicochemical and spectroscopic studies revealed that phosphate appeared to bind predominantly with the polysaccharide fraction, and to a relatively lesser extent to protein fraction of EBP. The electrostatic interactions with amino groups and ligand exchange with hydroxyl groups of EBP were found to be primary basis for phosphate binding mechanism. The results of this study implicate the feasibility of the EBP for commercial bioremediation processes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A. calcoaceticus-A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI-TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI-TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI-TOF mass spectra., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Structure-based rational mutagenesis for engineering protein functionality has been limited by the scarcity and difficulty of obtaining crystal structures of desired proteins. On the other hand, when high-throughput selection is possible, directed evolution-based approaches for gaining protein functionalities have been random and fortuitous with limited rationalization. We combine comparative modeling of dimer structures, ab initio loop reconstruction, and ligand docking to select positions for mutagenesis to create a library focused on the ligand-contacting residues. The rationally reduced library requirement enabled conservative control of the substitutions by oligonucleotide synthesis and bounding its size within practical transformation efficiencies (∼ 10(7) variants). This rational approach was successfully applied on an inducer-binding domain of an Acinetobacter transcription factor (TF), pobR, which shows high specificity for natural effector molecule, 4-hydroxy benzoate (4HB), but no native response to 3,4-dihydroxy benzoate (34DHB). Selection for mutants with high transcriptional induction by 34DHB was carried out at the single-cell level under flow cytometry (via green fluorescent protein expression under the control of pobR promoter). Critically, this selection protocol allows both selection for induction and rejection of constitutively active mutants. In addition to gain-of-function for 34DHB induction, the selected mutants also showed enhanced sensitivity and response for 4HB (native inducer) while no sensitivity was observed for a non-targeted but chemically similar molecule, 2-hydroxy benzoate (2HB). This is unique application of the Rosetta modeling protocols for library design to engineer a TF. Our approach extends applicability of the Rosetta redesign protocol into regimes without a priori precision structural information., (© 2015 Wiley Periodicals, Inc.)

The bacterionanofiber protein AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5, is responsible for the nonspecific, high adhesiveness and autoagglutination of this strain. Previously, we introduced the ataA gene into the nonadhesive Acinetobacter strain ST-550, which conferred high adhesiveness to this strain, immobilized its cells, and improved indigo productivity due to enhanced tolerance to the toxic substrate. In this study, we again demonstrated the effectiveness of this new microbial immobilization method using AtaA in a number of conditions. AtaA enabled the effective immobilization of growing, resting, and lyophilized cells of a type strain of Acinetobacter, ADP1, which is also intrinsically nonadhesive, onto the surface of several kinds of support ranging from artificial to natural materials and from hydrophobic polyurethane to hydrophilic glass. Immobilization with AtaA enabled exclusive cell growth in the support space and only a few cells existed in the bulk medium. Immobilization of resting cells drastically increased cell concentration, depending on the support material; dry cells of approximately 110 g/L could be immobilized onto glass wool. Finally, we demonstrated that ADP1 cells immobilized on polyurethane foam can undergo at least 10 repetitive reactions without inactivation during a 5-h period. Even after drying and storing for 3 days, the immobilized cells showed enzymatic activity and an ester hydrolysis reaction was repeated by simply transferring the support with the cells into a fresh reaction buffer.

A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A K M of 2.6 mM and a k cat of 0.26 s(-1) were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.

Efficiency of MALDI mass spectrometry for differentiation between phenotypic phase variants (in colony morphology and virulence/avirulence) was investigated.for saprotrophic and opportunistically pathogenic bacteria of five genera (Acinetobacter, Arthrobacter, Rhodococcus, Corynebacterium, and Escherichia). Analysis of MALDI spectra (on the SA and HCCA matrices) included: (1) determination of similarity of the protein spectra as a percentage of the common protein peaks to the total amount of proteins, which reflects the phylogenetic relationships of the objects and has been recommended for identification of closely related species; (2) comparison of intensities of the common peaks; and (3) the presence of specific peaks as determinative characteristics of the variants. Under the standard analytical conditions the similarity between the MALDI profiles was shown to increase in the row: genus-species-strain-variant. Assessment of intensities of the common peaks was most applicable for differentiation between phase variants, especially in the case of high similarity of their profiles. Phase variants (A. oxydans strain K14) with similar colony morphotypes (S, R, M, and S(m)) grown on different media (LB agar, TSA, and TGYg) exhibited differences in their protein profiles reflecting the differences in their physiological characteristics. This finding is in agreement with our previous results on screening of the R. opacus with similar colony morphology and different substrate specificity in decomposition of chlorinated phenols. Analysis of MALDI spectra is probably the only efficient method for detection of such variants.

A whole-cell bioreporter, Acinetobacter baylyi ADPWH&#95;recA, was used to estimate the genotoxicity and bioavailability of chromium (VI) [Cr(VI)] in contaminated soils. Upon direct exposure to pre-sonicated soil samples, ADPWH&#95;recA gave the highest response to the genotoxicity of Cr(VI) within 5 h with a detection limit of 2 µM Cr(VI). Investigations on sites contaminated with Cr(VI) revealed that soil-associated Cr(VI) was bioavailable to the bioreporter although it could not be extracted into the aqueous phase. The physical and chemical properties of soil might influence the bioavailability of Cr(VI), and higher genotoxicity was found in soils with a lower pH. This whole cell bioreporter approach makes it feasible to evaluate the bioavailability and genotoxicity of Cr(VI)-contaminated soils to uncover their potential impact on human health.

Strain A2 with algicidal activity against Microcystis aeruginosa was isolated and identified with the genus Acinetobacter on the basis of phenotypic tests and 16S rRNA gene analysis. It was identified with the species Acinetobactor guillouiae by partial rpoB sequence analysis. When 10% (v/v) of the bacterial culture was co-incubated with M. aeruginosa culture, algicidal efficiency reached 91.6% after 7 days. Supernatant of A2 culture showed similar algicidal activity, while the cell pellet had little activity, suggesting that Acinetobacter guillouiae A2 indirectly attacked M. aeruginosa cells by secreting an extracellular algicidal compound, which was characterized as heat-stable. A significant decrease in the microcystin (microcystin-LR) concentration was observed after 10% (v/v) addition of A2 culture. Transcription of three microcystin-related genes (mcyA, mcyD and mcyH) was also found to be inhibited. The algicidal compound 4-hydroxyphenethylamine was obtained by further isolation and purification using various chromatographic techniques. The EC50, 3d and EC50, 7d values of 4-hydroxyphenethylamine against M. aeruginosa were 22.5 and 10.3 mgL(-1), respectively. These results indicate that A. guillouiae strain A2 inhibits growth of M. aeruginosa and degrades microcystin production. The identified compound, 4-hydroxyphenethylamine, has potential for development as a new algicidal formulation or product., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Resistance of bacterial pathogens toward antibiotics has revived interest in lipopolysaccharide (LPS) motifs as potential therapeutic targets. The LPS of several pathogenic Acinetobacter strains comprises a 4,5-branched Kdo trisaccharide containing an uncommon (2→5)-linkage. In this contribution the first stereoselective glycosylation method for obtaining an α-Kdo-(2→5)-α-Kdo disaccharide in good yield is highlighted. The synthetic approach used for accessing this linkage type will allow for future studies of the immunoreactivity associated with this unique bacterial Kdo inner core structure.

We investigated the taxonomic status of a phenetically unique group of 25 Acinetobacter strains which were isolated from multiple soil and water samples collected in natural ecosystems in the Czech Republic. Based on the comparative sequence analyses of the rpoB, gyrB, and 16S rRNA genes, the strains formed a coherent and well separated branch within the genus Acinetobacter. The genomic uniqueness of the group at the species level was supported by the low average nucleotide identity values (≤77.37%) between the whole genome sequences of strain ANC 3994(T) (NCBI accession no. APOH00000000) and the representatives of the known Acinetobacter species. Moreover, all 25 strains created a tight cluster clearly separated from all hitherto described species based on whole-cell protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and shared a unique combination of metabolic and physiological properties. The capacity to assimilate l-histidine and the inability to grow at 35°C differentiated them from their phenotypically closest neighbor, Acinetobacter johnsonii. We conclude that the 25 strains represent a novel Acinetobacter species, for which the name Acinetobacter bohemicus sp. nov. is proposed. The type strain of A. bohemicus is ANC 3994(T) (=CIP 110496(T)=CCUG 63842(T)=CCM 8462(T))., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Gold nanoparticles have enormous applications in cancer treatment, drug delivery and nanobiosensor due to their biocompatibility. Biological route of synthesis of metal nanoparticles are cost effective and eco-friendly. Acinetobacter sp. SW 30 isolated from activated sewage sludge produced cell bound as well as intracellular gold nanoparticles when challenged with HAuCl4 salt solution. We first time report the optimization of various physiological parameters such as age of culture, cell density and physicochemical parameters viz HAuCl4 concentration, temperature and pH which influence the synthesis of gold nanoparticles. Gold nanoparticles thus produced were characterized by various analytical techniques viz. UV-Visible spectroscopy, X-ray diffraction, cyclic voltammetry, transmission electron microscopy, selected area electron diffraction, high resolution transmission electron microscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy and dynamic light scattering. Polyhedral gold nanoparticles of size 20 ± 10 nm were synthesized by 24 h grown culture of cell density 2.4 × 10(9) cfu/ml at 50 °C and pH 9 in 0.5 mM HAuCl4. It was found that most of the gold nanoparticles were released into solution from bacterial cell surface of Acinetobacter sp. at pH 9 and 50 °C.

Here we report a case of carbapenem resistance in a human clinical isolate that was found to be closely related to the newly described environmental species Acinetobacter indicus. This strain harboured the blaOXA-23 carbapenemase gene located on a conjugative plasmid. Partial sequencing of 16S rDNA and rpoB genes, together with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis, showed that this strain was distantly related to the Acinetobacter baumannii-calcoaceticus complex and was closely related to A. indicus., (Copyright © 2014. Published by Elsevier B.V.)

Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically relevant members of the Acinetobacter calcoaceticus-A. baumannii (Acb) complex and important nosocomial pathogens. These three species are genetically closely related and phenotypically similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this study, we investigated the use of a multiplex PCR-based assay designed to detect internal fragments of the 16S-23S rRNA intergenic region and the gyrB and recA genes. The assay was capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23 different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately identified clinically relevant Acb complex species except those 'between 1 and 3' or 'close to 13TU'. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex identification. Our results show that a single multiplex PCR assay can reliably differentiate clinically relevant Acb complex species. Thus, this method may be used to better understand the clinical differences between infections caused by these species., (© 2014 The Authors.)

A novel Gram-negative, obligate aerobic, non-motile, and both coccobacillus- and bacillus-shaped bacterium, designated strain HYN18(T), was isolated from the intestinal tract of a honey bee (Apis mellifera). The isolate was oxidase-negative and catalase-positive. Strain HYN18(T) showed optimum growth at 25°C, pH 6-7, and in the presence of 1% (w/v) NaCl in trypticase soy broth medium. The isolate was negative for hydrolyses of starch, casein, gelatin and urea, indole production from tryptone and hemolysis on sheep blood agar. A phylogenetic analysis based on the 16S rRNA gene and rpoB gene sequence showed that strain HYN18(T) was most closely related to Acinetobacter nectaris SAP 763.2(T) and A. boissieri SAP 284.1(T) with 98.3% and 98.1% similarity (16S rRNA gene), respectively, and 84.4% similarity with Acinetobacter nectaris SAP 763.2(T) (rpoB gene). The major cellular fatty acids were summed features 3 (comprising C16:1ω7c /C16:1ω6c ), C12:0 and C16:0. The main isoprenoid quinone was ubiquinone-9 (Q-9). The polar lipids of strain HYN18(T) were phosphatidylethanolamine, three unidentified lipids, an unidentified phospholipid and an unidentified glycolipid. The DNA G+C content was 40.6 mol%. DNA-DNA hybridization experiments indicated less than 33 ± 10% relatedness to the closest phylogenetic species, Acinetobacter nectaris SAP 763.2(T). Thus, the phenotypic, phylogenetic and genotypic analyses indicate that strain HYN18(T) is a novel species within the genus Acinetobacter, for which the name Acinetobacter apis is proposed. The type strain is HYN18(T) (=KACC 16906(T) =JCM 18575(T)).

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper) was able to accurately identify 98.6% (142/144) of Acinetobacter baumannii isolates, 72.4% (63/87) of A. nosocomialis isolates, and 97.6% (41/42) of A. pittii isolates. All Acinetobacter junii, A. ursingii, A. johnsonii, and A. radioresistens isolates (n = 28) could also be identified correctly by Bruker Biotyper., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)