Acinetobacter Baylyi Strain ADP1 Research Articles

Biosynthesis of the fatty acid isopropyl esters by engineered Escherichia coli.

The fatty acid methyl esters and fatty acid ethyl esters are known as biodiesels which are considered to be renewable, nontoxic and biodegradable biofuels. However, the conventional biodiesels show a high crystallization temperature which is one of the most critical obstacles against the widespread biodiesel usage. The high crystallization temperature of biodiesel can be reduced by replacing the methyl or ethyl ester with an isopropyl moiety. Here we report on a strategy to establish biosynthesis of the fatty acid isopropyl esters(FAIPEs) from the simple substrate glucose in Escherichia coli with heterologous coexpression of atoB encoded acetyl-CoA acetyltransferase and atoAD encode acetoacetyl-CoA transferase from E. coli, ADC encode acetoacetate decarboxylase from Clostridium acetobutylicum, ADH encoded NADP-dependent alcohol dehydrogenase from Clostridium beijerinckii, 'TesA encoded a truncated fatty acyl-ACP thioesterase and FadD encoded fatty acyl-CoA synthetase from E. coli, and the WS/DGAT encoded acyltransferase from Acinetobacter baylyi strain ADP1. It was found that the yield of FAIPEs was up to 203.4mg/L and accounted for around 6.4% (wt/wt) of the dry cell weight. Our results indicates that it is a feasible strategy to improve the yield of FAIPEs by increasing fatty acyl-CoA availability in biosynthetic pathway and exhibit a promising method for production of biodiesels with good low-temperature flow properties.

A whole-cell bioreporter approach for the genotoxicity assessment of bioavailability of toxic compounds in contaminated soil in China

A whole-cell bacterial bioreporter Acinetobacter baylyi strain ADP1_recA_lux that responds to genotoxins was employed to directly assess the adverse effects of the bioavailable fraction of mitomycin C (MMC), benzo[a]pyrene (BaP), chromium (VI) and lead (II) in amended soils and soil samples from two fragile areas in China without soil pre-treatment. The amended soils containing pollutants with the concentrations as low as 0.4 mg/kg MMC, 0.5 mg/kg BaP, 520 mg/kg Cr (VI) and 2072 mg/kg Pb (II) were found to be toxic. Soil particle-associated pollutants accounted for 86%, 100%, 29%, and 92% of the genotoxicity in the MMC, BaP, Cr (VI), and Pb (II) amended soil, respectively. The soils from contaminated sites were also valid to be genotoxic. The results suggest both free and soil particle-associated pollutants are bioavailable to soil organisms and a solid-phase contact bioreporter assay to soil contamination could provide a rapid screening tool for environmental risk assessment.

Investigating the barrier that prevents penicillin catabolism genes from transferring from PE‐S1G‐1 to Acinetobacter baylyi strain ADP1 (735.5)

Previous research discovered hundreds of phylogenetically diverse soil bacteria with the capacity to grow on a wide range of antibiotics as their sole carbon source. Some of them are closely related to human pathogens. The purpose of this study is to evaluate whether there is a barrier that might prevent the penicillin catabolism genes from transferring from PE‐S1G‐1 to Acinetobacter baylyi strain ADP1. Due to its natural competence and its close relation to human pathogen Acinetobacter baumannii, ADP1 is an ideal model for this study. Natural transformation with genomic DNA of PE‐S1G‐1, phi29 elongation with primers to add short homologous DNA to the genomic DNA, and Sau3A genomic plasmid libraries were used to test this barrier. None of these methods mentioned above transformed ADP1 to be a strain with penicillin catabolism phenotype. Surprisingly, a recent research challenges the concept of bacteria subsisting on antibiotics and argues that the selective media used in the original study (SCS) contains 15 mg/L EDTA which could be the carbon source that sustains the growth of these “antibiotic‐eaters.” The hypothesis that these bacteria could subsist on EDTA is currently being tested.

Neutral lipid production in Alcanivorax borkumensis SK2 and other marine hydrocarbonoclastic bacteria

AbstractTriacylglycerols (TAG) and wax esters (WE) constitute together with polyhydroxyalkanoates (PHA) the major storage lipophilic compounds in prokaryotes. Recently, the production of neutral lipids such as TAG and WE has been reported in species of the genus Alcanivorax, which belongs to the group of obligate hydrocarbonoclastic bacteria (OHCB). We analyzed the production of such neutral lipids by different marine hydrocarbonoclastic bacteria growing on pyruvate or hexadecane as sole carbon source, and compared it to other bacteria such as Rhodococcus opacus strain PD630 and Acinetobacter baylyi strain ADP1, which are two well‐studied strains for production of neutral lipids. Alcanivorax borkumensis SK2 synthesized mainly TAG when cells are cultivated on pyruvate, while biosynthesis and accumulation of WE was mainly observed in cells growing on hexadecane. Alcanivorax jadensis T9 synthesized and accumulated mainly WE if cells were cultivated with hexadecane, while both TAG and WE were observed if cells were cultivated with pyruvate as sole carbon source, respectively. Predominantly production as well as export of WE was observed in Marinobacter hydrocarbonoclasticus SP17 growing on pyruvate or hexadecane as sole carbon source. The chemical structures of TAG and WE produced by A. borkumensis SK2 were analyzed by gas chromatography and/or mass spectrometry, and first studies to investigate the influence of different C/N ratio (7, 50 or 150) on the production of neutral lipids were performed.

Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to approximately 10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.

Analysis of neutral lipid biosynthesis in Streptomyces avermitilis MA-4680 and characterization of an acyltransferase involved herein

The physiology of lipid production in Streptomyces avermitilis MA-4680 with regard to the fatty acid composition of the accumulated lipids and their cellular distribution was analyzed. Cells were able to accumulate about ten to 30 lipid granules with diameters between 100 and 500 nm filling about 70-80% of the cell cytoplasm. Gas chromatography/mass spectrometry analyses of total cellular lipids and from isolated triacylglycerols (TAG) confirmed a similar fatty acid composition with a large portion of iso- and anteiso-methyl-branched fatty acids. De novo biosynthesis of wax esters (WE) appeared only during cocultivation on glucose and hexadecanol as carbon source. Homology alignments with the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; AtfA) from Acinetobacter baylyi strain ADP1 yielded one open reading frame in the genome databases of S. avermitilis MA-4680 referred to as SAV7256 with 25.3% homology. The highly conserved HHAxxDG active site motif found in AtfA, which is present in SAV7256, as well as the similar hydrophobicity profiles of AtfA and SAV7256 indicate a similar structure and function of both proteins. High acyl-CoA:diacylglycerol acyltransferase activity (DGAT; 143 pmol (mg min)(-1)) but low wax ester synthase activity (WS; 1.3 pmol (mg min)(-1)) were detected in crude extracts of S. avermitilis, which were consistent with the high TAG and negligible WE content of the cells. This indicates that TAG accumulation in S. avermitilis MA-4680 is mediated by the classical acyl-CoA-dependent DGAT pathway. Heterologous expression experiments in recombinant Escherichia coli BL21(DE3) demonstrated both WS and DGAT enzyme activity of SAV7256. Furthermore, substrate specificities of the acyltransferase SAV7256 will be discussed.

Both histidine residues of the conserved HHXXXDG motif are essential for wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase catalysis

AbstractBacterial acyltransferases of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) family possess a highly conserved HHXXXDG motif. In this study, we describe the first experimental evidence that this motif is part of the active site of WS/DGAT from the Acinetobacter baylyi strain ADP1 and that it is crucial for enzymatic activity. The second histidine residue of this motif (H133) turned out to be essential for the catalytic activity. In addition, the replacement of the first histidine (His132) also led to explicitly decreased activity. A complete loss of activity was only observed upon substitution of both histidine residues by leucine, revealing that both are necessary for maximal activity. In contrast, the replacement of Asp137 and Gly138 against alanine had only little effect on enzyme activity, thus demonstrating that they are not essential for WS/DGAT catalysis although belonging to the highly conserved motif. One peculiarity of WS/DGAT enzymes is their little substrate specificity regarding hydrophobic compounds. In this study, we demonstrated the inability of WS/DGAT to accept polar compounds as substrates.

Cloning and characterization of a gene involved in triacylglycerol biosynthesis and identification of additional homologous genes in the oleaginous bacterium Rhodococcus opacus PD630

The oleaginous bacterium Rhodococus opacus strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of Acinetobacter baylyi strain ADP1 and Mycobacterium smegmatis mc(2)155. The atf fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete atf1 gene. An atf1 disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant. RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of atf1 under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from Rhodococcus jostii RHA1 sequence data was used. This yielded nine additional atf-homologous genes exhibiting 88-99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in Escherichia coli.

Linear chromosomes in bacteria: no straight edge advantage?

The beginning of 2007 brought us the complete genome of the yeast Pichia stipitis, five archaeal and more than 25 completely sequenced bacterial genomes (Table 1). In addition, there were two genomics-related papers that deserve a special discussion. One of them (Fuchs et al., 2007) described an unfinished genome of the ubiquitous marine phototrophic bacterium Congregibacter litoralis, while the other (Cui et al., 2007) examined the properties of the Escherichia coli K-12 strains with a linear chromosome.

Rapid Evolution of Diminished Transformability in Acinetobacter baylyi

The reason for genetic exchange remains a crucial question in evolutionary biology. Acinetobacter baylyi strain ADP1 is a highly competent and recombinogenic bacterium. We compared the parallel evolution of wild-type and engineered noncompetent lineages of A. baylyi in the laboratory. If transformability were to result in an evolutionary benefit, it was expected that competent lineages would adapt more rapidly than noncompetent lineages. Instead, regardless of competency, lineages adapted to the same extent under several laboratory conditions. Furthermore, competent lineages repeatedly evolved a much lower level of transformability. The loss of competency may be due to a selective advantage or the irreversible transfer of loss-of-function alleles of genes required for transformation within the competent population.

OPPORTUNITIES FOR GENETIC INVESTIGATION AFFORDED BYACINETOBACTER BAYLYI, A NUTRITIONALLY VERSATILE BACTERIAL SPECIES THAT IS HIGHLY COMPETENT FOR NATURAL TRANSFORMATION

The genetic and physiological properties of Acinetobacter baylyi strain ADP1 make it an inviting subject for investigation of the properties underlying its nutritional versatility. The organism possesses a relatively small genome in which genes for most catabolic functions are clustered in several genetic islands that, unlike pathogenicity islands, give little evidence of horizontal transfer. Coupling mutagenic polymerase chain reaction to natural transformation provides insight into how structure influences function in transporters, transcriptional regulators, and enzymes. With appropriate selection, mutants in which such molecules have acquired novel function may be obtained. The extraordinary competence of A. baylyi for natural transformation and the ease with which it expresses heterologous genes make it a promising platform for construction of novel metabolic systems. Steps toward this goal should take into account the complexity of existing pathways in which transmembrane trafficking plays a significant role.