Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Identification and characterization of the capsule depolymerase Dpo27 from phage IME-Ap7 specific to Acinetobacter pittii.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

Carbapenemase genes in clinical and environmental isolates of Acinetobacter spp. from Quito, Ecuador.

Carbapenem-resistant Acinetobacter spp. is associated with nosocomial infections in intensive care unit patients, resulting in high mortality. Although Acinetobacter spp. represent a serious public health problem worldwide, there are a few studies related to the presence of carbapenemases in health care facilities and other environmental settings in Ecuador. The main aim of this study was to characterize the carbapenem-resistant Acinetobacter spp. isolates obtained from four hospitals (52) and from five rivers (27) close to Quito. We used the disc diffusion and EDTA sinergy tests to determine the antimicrobial susceptibility and the production of metallo ß-lactamases, respectively. We carried out a multiplex PCR of gyrB gene and the sequencing of partial rpoB gene to bacterial species identification. We performed molecular screening of nine carbapenem-resistant genes (blaSPM, blaSIM, blaGIM, blaGES, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, and blaOXA-143) by multiplex PCR, followed by identification using sequencing of blaOXA genes. Our findings showed that carbapenem-resistant A. baumannii were the main species found in health care facilities and rivers. Most of the clinical isolates came from respiratory tract samples and harbored blaOXA-23, blaOXA-366, blaOXA-72, blaOXA-65, blaOXA-70, and blaOXA-143-like genes. The river isolates harbored only the blaOXA-51 and probably blaOXA-259 genes. We concluded that the most predominant type of carbapenem genes among isolates were both blaOXA-23 and blaOXA-65 among A. baumannii clinical isolates.

Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor.

Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species.

Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D ß-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most ß-lactam antibiotics in the fight against these resistant isolates.

A Multi-enzyme Cascade for the Biosynthesis of AICA Ribonucleoside Di- and Triphosphate.

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.

Characterization of a Stable Form of Carboxypeptidase G2 (Glucarpidase), a Potential Biobetter Variant, From Acinetobacter sp. 263903-1.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli.

Cholesterol oxidases (CHOXs) are flavin-adenine dinucleotide-dependent oxidoreductases with a range of biotechnological applications. There remains an urgent need to identify novel CHOX family members to meet the demands of enzyme markets worldwide. Here, we report the cloning, heterologous expression, and structural modeling of the cholesterol oxidase of Acinetobacter sp. strain RAMD. The cholesterol oxidase gene was cloned and expressed in pGEM®-T and pET-28a(+) vectors, respectively, using a gene-specific primer based on the putative cholesterol oxidase ORF of Acinetobacter baumannii strain AB030 (GenBank [gb] locus tag: IX87_05230). The obtained nucleotide sequence (1671 bp, gb: MK575469.2), translated to a protein designated choxAB (556 amino acids), was overexpressed as inclusion bodies (IBs) (MW ˜ 62 kDa) in 1 mm IPTG-induced Escherichia coli BL21 (DE3) Rosetta cells. The optimized expression conditions (1 mm IPTG with 2% [v/v] glycerol and at room temperature) yielded soluble active choxAB of 0.45 U·mL-1 , with 56.25-fold enhancement. The recombinant choxAB was purified to homogeneity using Ni2+ -affinity agarose column with specific activity (0.054 U·mg-1 ), yield (8.1%), and fold purification (11.69). Capillary isoelectric-focusing indicated pI of 8.77 for choxAB. LC-MS/MS confirmed the IBs (62 kDa), with 82.6% of the covered sequence being exclusive to A. baumannii cholesterol oxidase (UniProtKB: A0A0E1FG24). The 3D structure of choxAB was predicted using the LOMETS webtool with the cholesterol oxidase template of Streptomyces sp. SA-COO (PDB: 2GEW). The predicted secondary structure included 18 α-helices and 12 ß-strands, a predicted catalytic triad (E220 , H380 , and N514 ), and a conserved FAD-binding sequence (GSGFGGSVSACRLTEKG). Future studies should consider fusion to solubilization tags and switching to the expression host Pichia pastoris to reduce IB formation.

A Multi-Enzyme Cascade Reaction for the Production of 2'3'-cGAMP.

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2'3'-cGAMP) formation was successfully developed. The 2'3'-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2'3'-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2'3'-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.

Characterization and rational modification of aspartate 4-decarboxylase from Acinetobacter radioresistens for the production of l-alanine.

Enzymatic synthesis of l-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and l-alanine production because it catalyzes the decarboxylation of l-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5. Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of l-aspartic acid to l-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to l-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the l-alanine production industry, especially in the case of high optical purity requirements.

Structure and functional properties of the cold-adapted catalase from Acinetobacter sp. Ver3 native to the Atacama plateau in northern Argentina.

Heme catalases remove hydrogen peroxide by catalyzing its dismutation into water and molecular oxygen, thereby protecting the cell from oxidative damage. The Atacama plateau in northern Argentina, located 4000 m above sea level, is a desert area characterized by extreme UV radiation, high salinity and a large temperature variation between day and night. Here, the heme catalase KatE1 from an Atacama Acinetobacter sp. isolate was cloned, expressed and purified, with the aim of investigating its extremophilic properties. Kinetic and stability assays indicate that KatE1 is maximally active at 50°C in alkaline media, with a nearly unchanged specific activity between 0°C and 40°C in the pH range 5.5-11.0. In addition, its three-dimensional crystallographic structure was solved, revealing minimal structural differences compared with its mesophilic and thermophilic analogues, except for a conserved methionine residue on the distal heme side, which is proposed to comprise a molecular adaptation to oxidative damage.

The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

BACKGROUND: Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance. METHODS: A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN. RESULTS: MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants. CONCLUSIONS: Our investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Metallo-Beta-Lactamase-Producing Acinetobacter spp. from Clinical Isolates at a Tertiary Care Hospital in Ghana.

Metallo-beta-lactamase-producing Acinetobacter spp. is a major challenge for therapeutic treatment of nosocomial infections. This study is aimed at determining the prevalence of MBL-producing Acinetobacter spp. among 87 clinical isolates of Acinetobacter spp. from the Korle-Bu Teaching Hospital, Accra, between August 2014 and July 2015. Acinetobacter spp. was identified by standard bacteriological method, and resistance to different antibiotics was assessed with the Kirby-Bauer disc diffusion method. Meropenem-resistant Acinetobacter isolates were screened for enzyme activity using the modified Hodge test (MHT) and combined disc test (CDT). Additionally, multiplex PCR was used to determine MBL genes presence (blaVIM, blaIMP, and blaNDM). All Acinetobacter isolates showed high resistance to cefotaxime (90.8%), ceftazidime (75.9%), cotrimoxazole (70.1%), ciprofloxacin (64.4%), gentamicin (72.4%), levofloxacin (67.8%), and meropenem (59.8%). A total of 54 (62.1%) of Acinetobacter isolates were multidrug-resistant. Out of 52 (59.8%) meropenem-resistant Acinetobacter, 3 (5.8%) were carbapenemase producers by MHT, whilst, 23 (44.2%) were CDT positive. There was no significant difference between the resistance pattern of amikacin, ceftazidime, cotrimoxazole, ciprofloxacin, and meropenem amongst CDT-positive and CDT-negative isolates (p > 0.05). A total of 7/87 (8.1%) CDT-positive Acinetobacter isolates harboured blaNDM; of these, 4 (57.1%) were from wound swabs, urine (n = 2) (28.6%), and ear swab (n = 1) (14.3%). The study revealed that less than 9% of Acinetobacter spp. contained blaNDM encoding genes. Strict antibiotics usage plan and infection control measures are required to prevent the spread of these resistance genes.

The Glycoprotease CpaA Secreted by Medically Relevant Acinetobacter Species Targets Multiple O-Linked Host Glycoproteins.

Glycans decorate proteins and affect their biological function, including protection against proteolytic degradation. However, pathogenic, and commensal bacteria have evolved specific glycoproteases that overcome the steric impediment posed by carbohydrates, cleaving glycoproteins precisely at their glycosylation site(s). Medically relevant Acinetobacter strains employ their type II secretion system (T2SS) to secrete the glycoprotease CpaA, which contributes to virulence. Previously, CpaA was shown to cleave two O-linked glycoproteins, factors V and XII, leading to reduced blood coagulation. In this work, we show that CpaA cleaves a broader range of O-linked human glycoproteins, including several glycoproteins involved in complement activation, such as CD55 and CD46. However, only CD55 was removed from the cell surface, while CD46 remained unaltered during the Acinetobacter nosocomialis infection assay. We show that CpaA has a unique consensus target sequence that consists of a glycosylated serine or threonine residue after a proline residue (P-S/T), and its activity is not affected by sialic acids. Molecular modeling and mutagenesis analysis of CpaA suggest that the indole ring of Trp493 and the ring of the Pro residue in the substrate form a key interaction that contributes to CpaA sequence selectivity. Similar bacterial glycoproteases have recently gained attention as tools for proteomic analysis of human glycoproteins, and CpaA appears to be a robust and attractive new component of the glycoproteomics toolbox. Combined, our work provides insight into the function and possible application of CpaA, a member of a widespread class of broad-spectrum bacterial glycoproteases involved in host-pathogen interactions.IMPORTANCE CpaA is a glycoprotease expressed by members of the Acinetobacter baumannii-calcoaceticus complex, and it is the first bona fide secreted virulence factor identified in these species. Here, we show that CpaA cleaves multiple targets precisely at O-glycosylation sites preceded by a Pro residue. This feature, together with the observation that sialic acid does not impact CpaA activity, makes this enzyme an attractive tool for the analysis of O-linked human protein for biotechnical and diagnostic purposes. Previous work identified proteins involved in blood coagulation as targets of CpaA. Our work broadens the set of targets of CpaA, pointing toward additional roles in bacterium-host interactions. We propose that CpaA belongs to an expanding class of functionally defined glycoproteases that targets multiple O-linked host glycoproteins.

Protective Role of Bacterial Alkanesulfonate Monooxygenase under Oxidative Stress.

Bacterial alkane metabolism is associated with a number of cellular stresses, including membrane stress and oxidative stress, and the limited uptake of charged ions such as sulfate. In the present study, the genes ssuD and tauD in Acinetobacter oleivorans DR1 cells, which encode an alkanesulfonate monooxygenase and a taurine dioxygenase, respectively, were found to be responsible for hexadecanesulfonate (C16SO3H) and taurine metabolism, and Cbl was experimentally identified as a potential regulator of ssuD and tauD expression. The expression of ssuD and tauD occurred under sulfate-limited conditions generated during n-hexadecane degradation. Interestingly, expression analysis and knockout experiments suggested that both genes are required to protect cells against oxidative stress, including that generated by n-hexadecane degradation and H2O2 exposure. Measurable levels of intracellular hexadecanesulfonate were also produced during n-hexadecane degradation. Phylogenetic analysis suggested that ssuD and tauD are mainly present in soil-dwelling aerobes within the Betaproteobacteria and Gammaproteobacteria classes, which suggests that they function as controllers of the sulfur cycle and play a protective role against oxidative stress in sulfur-limited conditions.IMPORTANCEssuD and tauD, which play a role in the degradation of organosulfonate, were expressed during n-hexadecane metabolism and oxidative stress conditions in A. oleivorans DR1. Our study confirmed that hexadecanesulfonate was accidentally generated during bacterial n-hexadecane degradation in sulfate-limited conditions. Removal of this by-product by SsuD and TauD must be necessary for bacterial survival under oxidative stress generated during n-hexadecane degradation.

Quantitative and Qualitative Analyses of Triacylglycerol Production in the Wild-Type Cyanobacterium Synechocystis sp. PCC 6803 and the Strain Expressing AtfA from Acinetobacter baylyi ADP1.

Although cyanobacteria do not possess wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT), the bacterial enzyme for triacylglycerol (TAG) production, there have been several studies reporting the accumulation of TAG-like compounds in cyanobacteria. In this study, we aimed to evaluate TAG productivity of the ΔrecJ::atfA strain of Synechocystis sp. PCC 6803 generated by inserting atfA encoding WS/DGAT from Acinetobacter baylyi ADP1 into recJ (sll1354), together with the wild type (WT) and the gene-disrupted strain of slr2103 having homology with eukaryotic DGAT2 gene family (Δ2103). Thin-layer chromatography (TLC) of neutral lipids or isolation of the neutral lipid-enriched fraction followed by gas chromatography or liquid chromatography-tandem mass spectrometry was employed for analyses. The ΔrecJ::atfA strain accumulated 0.508 nmol ml-1OD730-1 of TAG after a week of incubation at 100 µmol photons m-2 s-1. The saturated fatty acids C16:0 and C18:0 accounted for about 50% and 20% of the TAG fatty acids, respectively, suggesting that de novo-synthesized fatty acids were preferentially incorporated into TAG molecules. When the neutral lipid profile of the lipid extracts was examined by TLC, a spot located in a slightly lower position compared with the TAG standard was detected in WT but not in the Δ2103 strain. TAG accumulation levels of both strains was only 0.01-0.03 nmol ml-1OD730-1, but the fatty acid composition was substantially different from that of the background. These results suggest that trace amounts of TAG can be produced in Synechocystis cells by enzymes other than Slr2103, and major constituents of the TAG-like spot are unknown lipid species produced by Slr2103.

Response of particle-associated bacteria to long-term heavy metal contamination in a tropical estuary.

Estuaries being the connecting link between terrestrial and marine environment, experience spatial variations in the hydrographic variables as well as concentrations of pollutants. The present study reports a contrasting difference in the metal tolerance and enzyme activity of particle-associated bacteria (PAB) isolated from the upstream and downstream reaches of a tropical estuary [Cochin Estuary (CE) in the southwest coast of India], exposed to different levels of heavy metal contamination. The upstream of the estuary has been overloaded with heavy metals in the last few decades, while the downstream is less polluted. There were only 25% of culturable PAB phylogenetically common in both upstream and downstream. The PAB isolated from the upstream were dominated by γ-proteobacteria (48.1%) followed by α-proteobacteria (25.0%), while it was in the reverse order of α-proteobacteria (45.9%) and γ-proteobacteria (36.1%) in the downstream. More number of PAB from the upstream showed tolerance to higher concentrations of Zn and Cd. The Acinetobacter sp. MMRF1051 isolated from the upstream showed tolerance up to 250 mM Zn, 100 mM Cd, and 250 mM Ni. The enzyme expression profile of PAB from downstream was in the order of lipase > phosphatase > ß-glucosidase > aminopeptidase, while it was in the order of ß-glucosidase > lipase > aminopeptidase > phosphatase in the upstream of the estuary. The present study shows the selective pressure exerted by heavy metal pollution on the diversity of culturable bacteria associated with particulate matter in a tropical estuary. Also, the variation in their enzyme activities may impinge the remineralization of particulate organic matter (POM) in the system and may impart adverse impacts on ecosystem functioning.

Characterization of a novel type I l-asparaginase from Acinetobacter soli and its ability to inhibit acrylamide formation in potato chips.

l-Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l-asparaginase was screened. Type I l-asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l-asparaginase had an activity of 42.0 IU mL-1 and showed no activity toward l-glutamine and d-asparagine. The recombinant l-asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and ß-mercaptoethanol. The Km and Vmax values of the l-asparaginase for the l-asparagine were 3.22 mmol L-1 and 1.55 IU µg-1, respectively. Moreover, the recombinant l-asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l-asparaginase has the potential for application in the food processing industry.

Catalases of the polyextremophylic Andean isolate Acinetobacter sp. Ver 3 confer adaptive response to H2 O2 and UV radiation.

The polyextremophilic strain Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes exhibits elevated tolerance to UV-B radiation and to pro-oxidants, a feature that has been correlated to its unusually high catalase activity. The Ver3 genome sequence analysis revealed the presence of two genes coding for monofunctional catalases: AV3 KatE1 and AV3 KatE2, the latter harboring an N-terminal signal peptide. We show herein that AV3 KatE1 displays one of the highest catalytic activities reported so far and is constitutively expressed at relatively high amounts in the cytosol, acting as the main protecting catalase against H2 O2 and UV-B radiation. The second catalase, AV3 KatE2, is a periplasmic enzyme strongly induced by both peroxide and UV, conferring supplementary protection against pro-oxidants. The N-terminal signal present in AV3 KatE2 was required not only for transport to the periplasm via the twin-arginine translocation pathway, but also for proper folding and subsequent catalytic activity. The analysis of catalase distribution among 114 Acinetobacter complete genomes revealed a great variability in the catalase classes, with A. baumannii clinical isolates exhibiting higher numbers of isoenzymes and the most variable profiles.