Acinetobacter Baylyi Research Articles

Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production

Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production

Plasmids affect microindel mutations in Acinetobacter baylyi ADP1

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can affect the level of microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid vector pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, results with the IncP plasmid vector pRK415 (theta replication mechanism) show that the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study (belonging to IncA/C2, IncW, pBBR incompatibility groups) confer ambiguous or no quantifiable mutagenic effects.

Natural Organic Matter Enhances Natural Transformation of Extracellular Antibiotic Resistance Genes in Sunlit Water.

Antibiotic resistance genes (ARGs) as emerging environmental contaminants exacerbate the risk of spreading antibiotic resistance. Natural organic matter (NOM) is ubiquitous in aquatic environments and plays a crucial role in biogeochemical cycles. However, its impact on the dissemination of extracellular antibiotic resistance genes (eARGs) under sunlight exposure remains elusive. This study reveals that environmentally relevant levels of NOM (0.1-20 mg/L) can significantly enhance the natural transformation frequency of the model bacterium Acinetobacter baylyi ADP1 by up to 7.6-fold under simulated sunlight. Similarly, this enhancement was consistently observed in natural water and wastewater systems. Further mechanism analysis revealed that reactive oxygen species (ROS) generated by NOM under sunlight irradiation, primarily singlet oxygen and hydroxyl radicals, play a crucial role in this process. These ROS induce intracellular oxidative stress and elevated cellular membrane permeability, thereby indirectly boosting ATP production and enhancing cell competence of extracellular DNA uptake and integration. Our findings highlight a previously underestimated role of natural factors in the dissemination of eARGs within aquatic ecosystems and deepen our understanding of the complex interplay between NOM, sunlight, and microbes in environmental water bodies. This underscores the importance of developing comprehensive strategies to mitigate the spread of antibiotic resistance in aquatic environments.

Evolution recovers the fitness of Acinetobacter baylyi strains with large deletions through mutations in deletion-specific targets and global post-transcriptional regulators.

Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-free Acinetobacter baylyi strain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulators rnd (ribonuclease D), csrA (RNA-binding carbon storage regulator), and hfq (RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restored csrA function. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments.

Protein overabundance is driven by growth robustness.

Protein expression levels optimize cell fitness: Too low an expression level of essential proteins will slow growth by compromising essential processes; whereas overexpression slows growth by increasing the metabolic load. This trade-off naïvely predicts that cells maximize their fitness by sufficiency, expressing just enough of each essential protein for function. We test this prediction in the naturally-competent bacterium Acinetobacter baylyi by characterizing the proliferation dynamics of essential-gene knockouts at a single-cell scale (by imaging) as well as at a genome-wide scale (by TFNseq). In these experiments, cells proliferate for multiple generations as target protein levels are diluted from their endogenous levels. This approach facilitates a proteome-scale analysis of protein overabundance. As predicted by the Robustness-Load Trade-Off (RLTO) model, we find that roughly 70% of essential proteins are overabundant and that overabundance increases as the expression level decreases, the signature prediction of the model. These results reveal that robustness plays a fundamental role in determining the expression levels of essential genes and that overabundance is a key mechanism for ensuring robust growth.

Initiation of H1-T6SS dueling between Pseudomonas aeruginosa.

The Type VI secretion system (T6SS) is a multicomponent apparatus, present in many Gram-negative bacteria, which can inhibit bacterial prey in various ecological niches. Pseudomonas aeruginosa assembles one of its three T6SS (H1-T6SS) to respond to attacks from adjacent competing bacteria. Surprisingly, repeated assemblies of the H1-T6SS, termed dueling, were described in a monoculture in the absence of an attacker strain; however, the underlying mechanism was unknown. Here, we explored the role of H2-T6SS of P. aeruginosa in triggering H1-T6SS assembly. We show that H2-T6SS inactivation in P. aeruginosa causes a significant reduction in H1-T6SS dueling and that H2-T6SS activity directly triggers retaliation by the H1-T6SS. Intraspecific competition experiments revealed that elimination of H2-T6SS in non-immune prey cells conferred protection from H1-T6SS. Moreover, we show that the H1-T6SS response is triggered independently of the characterized lipase effectors of the H2-T6SS, as well as those of Acinetobacter baylyi and Vibrio cholerae. Our results suggest that H1-T6SS response to H2-T6SS in P. aeruginosa can impact intraspecific competition, particularly when the H1-T6SS effector-immunity pairs differ between strains, and could determine the outcome of multistrain colonization.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa harbors three different Type VI secretion systems (H1, H2, and H3-T6SS), which can translocate toxins that can inhibit bacterial competitors or inflict damage to eukaryotic host cells. Unlike the unregulated T6SS assembly in other Gram-negative bacteria, the H1-T6SS in P. aeruginosa is precisely assembled as a response to various cell damaging attacks from neighboring bacterial cells. Surprisingly, it was observed that neighboring P. aeruginosa cells repeatedly assemble their H1-T6SS toward each other. Mechanisms triggering this "dueling" behavior between sister cells were unknown. In this report, we used a combination of microscopy, genetic and intraspecific competition experiments to show that H2-T6SS initiates H1-T6SS dueling. Our study highlights the interplay between different T6SS clusters in P. aeruginosa, which may influence the outcomes of multistrain competition in various ecological settings such as biofilm formation and colonization of cystic fibrosis lungs.

Prophage induction by non-antibiotic compounds promotes transformation of released antibiotic resistance genes from cell lysis

Prophages are prevalent among bacterial species, including strains carrying antibiotic resistance genes (ARGs). Prophage induction can be triggered by the SOS response to stressors, leading to cell lysis. In environments polluted by chemical stressors, ARGs and prophage co-harboring strains might pose an unknown risk of spreading ARGs through chemical pollutant-mediated prophage induction and subsequent cell lysis. In this study, we investigated the effects of common non-antibiotic water pollutants, triclosan and silver nanoparticles, on triggering prophage induction in clinical isolates carrying ARGs and the subsequent uptake of released ARGs by the naturally competent bacterium Acinetobacter baylyi. Our results demonstrate that both triclosan and silver nanoparticles, at environmentally relevant concentrations and those found in commercial products, significantly enhance prophage induction among various clinical isolates. Transmission electron microscopy imaging and plaque assays confirmed the production of infectious phage particles under non-antibiotic pollutants-mediated prophage induction. In addition, the rate of ARG transformation to A. baylyi significantly increased after the release of extracellular ARGs from prophage induction-mediated cell lysis. The mechanism of non-antibiotic pollutants-mediated prophage induction is primarily associated with excessive oxidative stress, which provokes the SOS response. Our findings offer insights into the role of non-antibiotic pollutants in promoting the dissemination of ARGs by triggering prophage induction.

Use of zebrafish to identify host responses specific to type VI secretion system mediated interbacterial antagonism.

Interbacterial competition is known to shape the microbial communities found in the host, however the interplay between this competition and host defense are less clear. Here, we use the zebrafish hindbrain ventricle (HBV) as an in vivo platform to investigate host responses to defined bacterial communities with distinct forms of interbacterial competition. We found that antibacterial activity of the type VI secretion system (T6SS) from both Vibrio cholerae and Acinetobacter baylyi can induce host inflammation and sensitize the host to infection independent of any individual effector. Chemical suppression of inflammation could resolve T6SS-dependent differences in host survival, but the mechanism by which this occurred differed between the two bacterial species. By contrast, colicin-mediated antagonism elicited by an avirulent strain of Shigella sonnei induced a negligible host response despite being a more potent bacterial killer, resulting in no impact on A. baylyi or V. cholerae virulence. Altogether, these results provide insight into how different modes of interbacterial competition in vivo affect the host in distinct ways.

Effect of chemotherapeutic agents on natural transformation frequency in Acinetobacter baylyi.

Natural transformation is the ability of a bacterial cell to take up extracellular DNA which is subsequently available for recombination into the chromosome (or maintenance as an extrachromosomal element). Like other mechanisms of horizontal gene transfer, natural transformation is a significant driver for the dissemination of antimicrobial resistance. Recent studies have shown that many pharmaceutical compounds such as antidepressants and anti-inflammatory drugs can upregulate transformation frequency in the model species Acinetobacter baylyi. Chemotherapeutic compounds have been shown to increase the abundance of antimicrobial resistance genes and increase colonization rates of potentially pathogenic bacteria in patient gastrointestinal tracts, indicating an increased risk of infection and providing a pool of pathogenicity or resistance genes for transformable commensal bacteria. We here test for the effect of six cancer chemotherapeutic compounds on A. baylyi natural transformation frequency, finding two compounds, docetaxel and daunorubicin, to significantly decrease transformation frequency, and daunorubicin to also decrease growth rate significantly. Enhancing our understanding of the effect of chemotherapeutic compounds on the frequency of natural transformation could aid in preventing the horizontal spread of antimicrobial resistance genes.

Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria

BackgroundLignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO2.ResultsIn this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO2 to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO2 and guaiacol as carbon sources.ConclusionsBy the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

DNA binding is rate-limiting for natural transformation.

Bacteria take up environmental DNA using dynamic appendages called type IV pili (T4P) to elicit horizontal gene transfer in a process called natural transformation. Natural transformation is widespread amongst bacteria yet determining how different factors universally contribute to or limit this process across species has remained challenging. Here we show that Acinetobacter baylyi, the most naturally transformable species, is highly transformable due to its ability to robustly bind nonspecific DNA via a dedicated orphan minor pilin, FimT. We show that, compared to its homologues, A. baylyi FimT contains multiple positively charged residues that additively promote DNA binding efficiency. Expression of A. baylyi FimT in a closely related Acinetobacter pathogen is sufficient to substantially improve its capacity for natural transformation, demonstrating that T4P-DNA binding is a rate-limiting step in this process. These results demonstrate the importance of T4P-DNA binding efficiency in driving natural transformation, establishing a key factor limiting horizontal gene transfer.

The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1.

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.

Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1

BackgroundLignocellulosic biomass as feedstock has a huge potential for biochemical production. Still, efficient utilization of hydrolysates derived from lignocellulose is challenged by their complex and heterogeneous composition and the presence of inhibitory compounds, such as furan aldehydes. Using microbial consortia where two specialized microbes complement each other could serve as a potential approach to improve the efficiency of lignocellulosic biomass upgrading.ResultsThis study describes the simultaneous inhibitor detoxification and production of lactic acid and wax esters from a synthetic lignocellulosic hydrolysate by a defined coculture of engineered Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. A. baylyi ADP1 showed efficient bioconversion of furan aldehydes present in the hydrolysate, namely furfural and 5-hydroxymethylfurfural, and did not compete for substrates with S. cerevisiae, highlighting its potential as a coculture partner. Furthermore, the remaining carbon sources and byproducts of S. cerevisiae were directed to wax ester production by A. baylyi ADP1. The lactic acid productivity of S. cerevisiae was improved approximately 1.5-fold (to 0.41 ± 0.08 g/L/h) in the coculture with A. baylyi ADP1, compared to a monoculture of S. cerevisiae.ConclusionThe coculture of yeast and bacterium was shown to improve the consumption of lignocellulosic substrates and the productivity of lactic acid from a synthetic lignocellulosic hydrolysate. The high detoxification capacity and the ability to produce high-value products by A. baylyi ADP1 demonstrates the strain to be a potential candidate for coculture to increase production efficiency and economics of S. cerevisiae fermentations.

Evaluation of antibacterial effects of stem extracts from Coscinium fenestratum.

An increasing number of pathogens are becoming antimicrobial resistant, among them are bacteria Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter species, Pseudomonas aeruginosa, known as ESKAPE pathogens. These ESKAPE pathogens have been cited as one of the leading causes of nosocomial infections around the world and have shown higher antimicrobial resistance posing a great clinical challenge and the need of new therapeutic agents. Various plant extracts have been used in traditional medicine since ancient times. Coscinium fenestratum, commonly called yellow vine is a vine native to South Asia and has been used to treat common cold, tetanus, fever, inflammation, wounds, and diabetes especially in countries such as Sri Lanka and India. In this study we extracted the secondary metabolites from dried C. fenestratum stem. The extract was then tested for antimicrobial properties using disc diffusion method. The bacteria assayed include Pseudomonas putida, Mycobacterium smegmatis, and Acinetobacter baylyi which are the safer relatives of P. aeruginosa, M. tuberculosis, and A. baumannii respectively. Methanolic extract of C. fenestratum at 100 µg did not show any antimicrobial activity against M. smegmatis. The effects of the extract will be tested further against P. putida and A. baylyi. If the tests show antimicrobial properties, we will determine the chemical composition of the methanolic extract revealing the chemical agent which is active on the bacteria. Our work will provide valuable information on potential use of C. fenestratum secondary metabolites in treating human pathogens contributing to the drug discovery pipeline.

Phthalates Boost Natural Transformation of Extracellular Antibiotic Resistance Genes through Enhancing Bacterial Motility and DNA Environmental Persistence.

The environmental dissemination of extracellular antibiotic resistance genes (eARGs) in wastewater and natural water bodies has aroused growing ecological concerns. The coexisting chemical pollutants in water are known to markedly affect the eARGs transfer behaviors of the environmental microbial community, but the detailed interactions and specific impacts remain elusive so far. Here, we revealed a concentration-dependent impact of dimethyl phthalate (DMP) and several other types of phthalate esters (common water pollutants released from plastics) on the natural transformation of eARGs. The DMP exposure at an environmentally relevant concentration (10 μg/L) resulted in a 4.8-times raised transformation frequency of Acinetobacter baylyi but severely suppressed the transformation at a high concentration (1000 μg/L). The promotion by low-concentration DMP was attributed to multiple mechanisms, including increased bacterial mobility and membrane permeability to facilitate eARGs uptake and improved resistance of the DMP-bounded eARGs (via noncovalent interaction) to enzymatic degradation (with suppressed DNase activity). Similar promoting effects of DMP on the eARGs transformation were also found in real wastewater and biofilm systems. In contrast, higher-concentration DMP suppressed the eARGs transformation by disrupting the DNA structure. Our findings highlight a potentially underestimated eARGs spreading in aquatic environments due to the impacts of coexisting chemical pollutants and deepen our understanding of the risks of biological-chemical combined pollution in wastewater and environmental water bodies.

Elongation Factor P is Required for Processes Associated with Acinetobacter Pathogenesis

Antibiotic resistance is one of the world’s fastest-growing and most prevalent problems today. The influx of antibiotics within our environment from inadequate antibiotic stewardship has led to a surge of drug-resistant microorganisms. The CDC has classified Carbapenem-resistant Acinetobacter (CRA) as an urgent threat within this crisis. New drug development is imperative to combat infections caused by drug-resistant pathogens such as CRA. Bacterial translation, the process of protein synthesis by the ribosome, is a common target for new antibiotic development. Elongation factor P (EF-P) is a universally conserved translation factor required for antibiotic resistance in many bacteria. In this study, we assessed the importance of EF-P in processes associated with Acinetobacter pathogenesis. In the absence of EF-P, Acinetobacter baylyi displays decreased biofilm formation, surface-associated motility, and resistance to beta-lactams and carbapenems. This data holds hope for future drug development targeting EF-P in pathogens closely related to A. baylyi. KEYWORDS: Acinetobacter baylyi; Translation; Ribosome; Elongation Factor P; Polyproline; Biofilm; Surface Associated Motility; Antibiotic Resistance

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

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

Regulation of tricarboxylate transport and metabolism in Acinetobacter baylyi ADP1.

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

Influence of furfural on the physiology of Acinetobacter baylyi ADP1.

Pretreatment of lignocellulosic biomass produces growth inhibitory substances such as furfural which is toxic to microorganisms. Acinetobacter baylyi ADP1 cannot use furfural as a carbon source, instead it biotransforms this compound into difurfuryl ether using the reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydrogenases AreB and FrmA during aerobic acetate catabolism. However, NADH consumption for furfural biotransformation compromises aerobic growth of A. baylyi ADP1. Depending on the growth phase, several genes related to acetate catabolism and oxidative phosphorylation changed their expression indicating that central metabolic pathways were affected by the presence of furfural. During the exponential growth phase, reactions involved in the formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (icd gene) and NADH (sfcA gene) were preferred when furfural was present. Therefore a higher NADH and NADPH production might support furfural biotransformation and biomass production, respectively. In contrast, in the stationary growth phase genes of the glyoxylate shunt were overexpressed probably to save carbon compounds for biomass formation, and only NADH regeneration was appreciated. Finally, disruption of the frmA or areB gene in A. baylyi ADP1 led to a decrease in growth adaptation and in the capacity to biotransform furfural. The characterization of this physiological behavior clarifies the impact of furfural in Acinetobacter metabolism.

AlmA involved in the long-chain n-alkane degradation pathway in Acinetobacter baylyi ADP1 is a Baeyer-Villiger monooxygenase.

Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26-C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10-C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer-Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.IMPORTANCEMany microbial studies on n-alkane degradation are focused on the genes involved in short-chain n-alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n-alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n-alkane degradation by Acinetobacter spp.; however, its role in the n-alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer-Villiger monooxygenase activity, thus indicating the presence of a novel LC n-alkane biodegradation mechanism in strain ADP1.