ackA Gene Research Articles

Microbial upcycling of methane to phytoene using metabolically engineered Methylocystis sp. MJC1 strain

Methane, a potent greenhouse gas, requires sustainable mitigation strategies. Here, the microbial upcycling of methane to phytoene, a valuable colorless carotenoid with applications in the cosmeceutical industry was demonstrated. To achieve this goal, a stepwise metabolic engineering approach was employed in Methylocystis sp. MJC1, a methane-oxidizing bacterium. The incorporation of crtE and crtB genes from Deinococcus radiodurans R1 established the phytoene biosynthetic pathway. This pathway was fine-tuned through promoter optimization, resulting in a phytoene production of 450 μg/L from 37 mmol/L methane. Disrupting the ackA gene reduced a by-product, acetate, by 50 % and increased phytoene production by 56 %. Furthermore, overexpressing the dxs gene boosted phytoene titer 3-fold. The optimized strain produced 15 mg/L phytoene from 2 mol/L methane in fed-batch fermentation, a 4-fold increase in phytoene titer and 4-fold in yield. This demonstrates Methylocystis sp. MJC1′s potential for efficient phytoene production and presents a novel approach for greenhouse gas reduction.

Effect and potential mechanism of nitrite reductase B on nitrite degradation by Limosilactobacillus fermentum RC4

Nitrite has the potential risk of hypoxic poisoning or cancer in pickled food. In our previous study, Limosilactobacillus fermentum (L. fermentum) RC4 is effective in nitrite degradation by producing nitrite reductase B (NirB). To investigate the detailed mechanism from the genome, response, and regulation of NirB, the whole-genome sequence of L. fermentum RC4 was analyzed, the L. fermentum-EGFP-nirB with enhanced green fluorescent protein (EGFP) labeled the nitrite reductase large subunit nirB, and the recombined L. fermentum-NirB with overexpression NirB strain was conducted. The key genes within the dominant metabolism pathways may be involved in stress tolerance to regulate the degrading process. The green fluorescence density of EGFP indicated that NirB activity has a threshold and peaked under 300 mg/L nitrite concentration. NirB overexpressed in L. fermentum RC4 boosted the enzyme activity by 39.6% and the degradation rate by 10.5%, when fermented in 300 mg/L for 40 h, compared to the control group. RNA-seq detected 248 differential genes mainly enriched in carbohydrate, amino acid, and energy metabolism. The ackA gene for pyruvate metabolism and the mtnN gene for cysteine metabolism were up-regulated. NirB regulates these genes to produce acid and improve stress resistance for L. fermentum RC4 to accelerate nitrite degradation.

New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

Succinic acid (SA), a C4 tricarboxylic acid cycle intermediate, is used as raw material for bulk chemicals and specialty chemicals, such as tetrahydrofuran and 1,4-butanediol, as well as also being used to synthesize the biodegradable biopolymers PBS (polymer poly (butylene succinate)). Actinobacillus succinogenes, which is facultative anaerobic and gram-negative, is one of the most promising natural SA-producing organisms, but genetic engineering of A. succinogenes is rare so far. In this study, a series of engineered strains was constructed using the pLGZ922 expression vector and a cytosine base editor (CBE) based on CRIPSR/Cas9; we found that phosphoenolpyruvate carboxylase (PEPC) was more important for the CO2 fixation pathway than pyruvate carboxylase (PYC) in A. succinogenes, and the annotated oxaloacetic acid decarboxylase (Asuc_0301 and Asuc_0302) had little correlation with the SA synthesis pathway. The by-product pathway was closely related to cell growth, and overexpression of FDH was beneficial to growth, while the knockout of the ackA gene reduced the growth. For the first time, the hypothetic sugars and SA transporters were mined and identified in A. succinogenes, of which Asuc_0914 was responsible for glucose uptake, and Asuc_0715 and Asuc_0716 constituted SA exporters. This deepens the understanding of SA biosynthesis in A. succinogenes and is also valuable for SA production by fermentation.

Elevated acetate kinase (ackA) gene expression, activity, and biofilm formation observed in methicillin-resistant strains of Staphylococcus aureus (MRSA)

BackgroundStaphylococcus aureus spreads its infections through biofilms. This usually happens in the stationary phase of S. aureus growth where it utilizes accumulated acetate as a carbon source via the phosphotrans-acetylase-acetate kinase (Pta-Ack) pathway. In which acetate kinase (ackA) catalyzes the substrate-level phosphorylation, a vital secondary energy-yielding pathway that promotes biofilms formation aids bacterium survival in hostile environments. In this study, we describe the cloning, sequencing, and expression of S. aureus ackA gene. The expression analysis of ackA gene in methicillin-resistant strains of S. aureus (MRSA) correlates with ackA activity and biofilm units. The uniqueness of ackA was analyzed by using in silico methods. ResultsElevated ackA gene expression was observed in MRSA strains, which correlates with increased ackA activity and biofilm units, explaining ackA role in MRSA growth and pathogenicity. The pure recombinant acetate kinase showed a molecular weight of 44 kDa, with enzyme activity of 3.35 ± 0.05 μM/ml/min. The presence of ACKA-1, ACKA-2 sites, one ATP, and five serine/threonine-protein kinase sites in the ackA gene (KC954623.1) indicated that acetyl phosphate production is strongly controlled. The comparative structural analysis of S. aureus ackA with ackA structures of Mycobacterium avium (3P4I) and Salmonella typhimurium (3SLC) exhibited variations as indicated by the RMSD values 1.877 Å and 2.141 Å respectively, explaining why ackA functions are differently placed in bacteria, concurring its involvement in S. aureus pathogenesis. ConclusionsOverall findings of this study highlight the correlation of ackA expression profoundly increases survival capacity through biofilm formation, which is a pathogenic factor in MRSA and plays a pivotal role in infection spreading.

Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes

Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.

Genomic and Biochemical Characterization of Bifidobacterium pseudocatenulatum JCLA3 Isolated from Human Intestine.

Bifidobacteria have been investigated due to their mutualistic microbe-host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5-5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3.

Enhancement of 2,3-Butanediol Production by Klebsiella pneumoniae: Emphasis on the Mediation of sRNA-SgrS on the Carbohydrate Utilization

The demand for renewable energy is increasing. Klebsiella pneumoniae is one of the most promising strains to produce 2,3-butanediol (2,3-BD). Compared with chemical methods, the biological production of 2,3-BD has the characteristics of substrate safety, low cost, and low energy consumption. However, excessive glucose concentrations can cause damage to cells. Therefore, this study investigated the effect of sRNA-SgrS as a sugar transport regulator on the fermentative production of 2,3-BD by K. pneumoniae in response to sugar stress. We designed multiple mutants of K. pneumoniae HD79 to redistribute its carbon flux to produce 2,3-BD. It was found that the 2,3-BD yield of sgrS overexpressed strain decreased by 44% compared with the original strain. The results showed that a high concentration of sRNA-SgrS could accelerate the degradation of ptsG mRNA (encoding the glucose transporter EIICBGlc) and downregulate the expression levels of the budA gene (encoding the α-acetyllactate decarboxylase) and the budB gene (encoding the α-acetyllactate synthase) and budC gene (encoding the 2,3-BD dehydrogenase) but had no effect on the ack gene (encoding the acetate kinase) and the ldh gene (encoding the lactate dehydrogenase). It provides a theoretical basis and a technical reference for understanding the complex regulation mechanism of sRNA in microorganisms and the genetics and breeding in industrial fermentation engineering.

Acetyl Metabolite Toxicity Impacts Bacillus subtilis Cell Growth and Development

Central carbon metabolism acts as a regulator of bacterial intracellular activity based on environmental conditions. Understanding the processes regulated by metabolism will provide insight into how bacteria grow and develop for applications in biotechnology and infectious disease. Acetyl-Coenzyme A (acetyl-CoA), a molecular intermediate of carbon metabolism, is essential for various cellular processes including energy production, cell development, and protein acetylation. The acetate pathway acts as a relief mechanism for excess carbon metabolism by converting acetyl-CoA into acetate, which is secreted by the cell. Inactivation of the acetate pathway causes a buildup of acetyl-CoA accompanied by delayed growth and cell death in Bacillus subtilis, a Gram-positive soil dwelling bacterium commonly used as a model for molecular microbiology research. However, the mechanism behind these acetyl-CoA-mediated growth defects are unknown. The acetate pathway was inactivated in B. subtilis by knocking out the ackA gene (ΔackA). ΔackA suppressor mutants were isolated based on recovered growth, and morphology studies and whole genome sequencing were performed to investigate underlying pathways. ΔackA cells exhibited altered morphology, including elongated cells and blebbing, which indicated defects in cell division and cell wall biosynthesis. The suppressors, however, exhibited significantly recovered growth and morphology resembling wild type cells. Whole genome sequencing revealed that several suppressors had mutations in pyruvate dehydrogenase complex genes pdhA and pdhC, likely inhibiting production of excess acetyl-CoA. An additional mutation was observed in dapH, a gene involved in the acetylation of a cell wall precursor. These results suggest that acetyl-CoA levels act as a signaling mechanism to connect metabolic status to cell wall biosynthesis and that the cell maintains a balance of acetyl metabolites for proper growth. This study deepens our understanding of metabolism as a regulatory mechanism for cellular processes.

Metal-organic frameworks coupling simultaneous saccharication and fermentation for enhanced butyric acid production from rice straw under visible light by Clostridium tyrobutyricum CtΔack::cat1

Here, Metal-Organic Frameworks (MOFs) coupling simultaneous saccharification and fermentation for butyric acid production using rice straw was constructed. Clostridium tyrobutyricum Δack::cat1, with deleted ack gene and overexpressed cat1 gene, was used as the butyric-acid-fermentation strain. MOFs was employed as a photocatalyst to improve butyric acid production, as well as a cytoprotective exoskeleton with immobilized cellulase for the hydrolysis of rice straw. Thus, the survival of MOFs-coated strain, the thermostability and pH stability of cellulase both remarkably increased. As a result, 55% of rice straw was hydrolyzed in 24 h, and the final concentration of butyric acid in visible light was increased by 14.23% and 29.16% compared to uncoated and coated strain without visible light, respectively. Finally, 26.25 g/L of butyric acid with a productivity of 0.41 g/L·h in fed-batch fermentation was obtained. This novel process inspires green approach of abundant low-cost feedstocks utilization for chemical production.

Acetate Kinase a Antisense Delivery by PAMAM Dendrimer for Decreasing Acetate Production and Increasing the Production of Recombinant Albumin in E. coli.

Background: Acetate accumulation in the culture medium is known as an inhibitor in recombinant protein production in Escherichia coli. Various approaches have been proposed and evaluated to overcome this challenge and reduce the concentration of acetate. In this study, we examined the effect of acetate kinase A antisense on acetate production rate in E. coli We also used PAMAM dendrimers as a suitable delivery agent for antisense transformation into E. coli host cell. Objective: This study aimed to decrease acetate production as a by-product using an antisense-dendrimer complex to increase mass cell and subsequently recombinant Albumin production in E. coli.Materials and Methods: Here, to study the effect of this treatment on recombinant protein production, we used pET22b/HAS construct. The ackA gene expression was inhibited by designed antisense to reduce acetate concentration in culture medium. AckA antisense was transferred to E. coli by PAMAM dendrimer. Finally, ackA expression and recombinant Albumin production were evaluated Real-Time PCR and densitometry, respectively.Results: Our data showed, designed antisense lead to reduction of acetate kinase gene expression and subsequently acetate concentration in the culture medium. Finally, acetate concentration reduction and cell mass increase result in enhanced recombinant Alb production in the treated group (1.25 mg.mL-1) compare to the control group ( 0.59 mg.mL-1).Conclusions: Reduction of acetate in E. coli fermentation process decreased the recombinant Alb production following cell growth and cell mass increase. In the current study, we showed that an antisense can be a useful tool for ackA gene expression reduction. Also, we noted that PAMAM dendrimer could be a proper delivery agent for oligonucleotide antisense transformation into bacterial cells.

Improvement of Streptococcus suis glutamate dehydrogenase expression in Escherichia coli through genetic modification of acetate synthesis pathway.

Escherichia coli generates acetate as an undesirable by-product that has several negative effects on protein expression, and the reduction of acetate accumulation by modifying genes of acetate synthesis pathway can improve the expression of recombinant proteins. In the present study, the effect of phosphotransacetylase (pta) or/and acetate kinase (ackA) deletion on glutamate dehydrogenase (GDH) expression was investigated. The results indicated that the disruptions of pta or/and ackA decreased the acetate accumulation and synthesis of per gram cell, and increased cell density, and GDH expression and synthesis of per gram cell. The pta gene was more important for acetate formation than the ackA gene. Using the strain with deletions of pta-ackA (SSGPA) for GDH expression, acetate accumulation (2·61gl-1 ) and acetate synthesis of per gram cell (0·229gg-1 ) were lowest, decreasing by 28·29 and 41·43% compared with those of the parental strain (SSG) respectively. The flux of acetate synthesis (6·6%) was decreased by 72·15% compared with that of SSG, and the highest cell density (11·38gl-1 ), GDH expression (2·78mgml-1 ), and GDH formation of per gram cell (0·2442mgmg-1 ) were obtained, which were 1·22-, 1·43- and 1·17-times higher than the parental strain respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Acetate is the key undesirable by-product in Escherichia coli cultivation, and both biomass and production of desired products are increased by the reduction of acetate accumulation. In the present study, the strains with deletions of pta or/and ackA were constructed to reduce the acetate accumulation and improve the GDH expression, and the highest expression level of GDH was obtained using the strain with lesion in pta-ackA that was 1·17-times higher than that of the parental strain. The construction strategy of recombinant E. coli for decreasing the acetate excretion can be used for high expression level of other desired products.

Haemophilus influenzae Glucose Catabolism Leading to Production of the Immunometabolite Acetate Has a Key Contribution to the Host Airway-Pathogen Interplay.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and impaired airway immunity, which provides an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. Clinical evidence supports that the COPD airways present increased concentrations of glucose, which may facilitate proliferation of pathogenic bacteria able to use glucose as a carbon source. NTHi metabolizes glucose through respiration-assisted fermentation, leading to the excretion of acetate, formate, and succinate. We hypothesized that such specialized glucose catabolism may be a pathoadaptive trait playing a pivotal role in the NTHi airway infection. To find out whether this is true, we engineered and characterized bacterial mutant strains impaired to produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA genes, respectively. While the inactivation of the pflA and frdA genes only had minimal physiological effects, the inactivation of the ackA gene affected acetate production and led to reduced bacterial growth, production of lactate under low oxygen tension, and bacterial attenuation in vivo. Moreover, bacterially produced acetate was able to stimulate the expression of inflammatory genes by cultured airway epithelial cells. These results back the notion that the COPD lung supports NTHi growth on glucose, enabling production of fermentative end products acting as immunometabolites at the site of infection. Thus, glucose catabolism may contribute not only to NTHi growth but also to bacterially driven airway inflammation. This information has important implications for developing nonantibiotic antimicrobials, given that airway glucose homeostasis modifying drugs could help prevent microbial infections associated with chronic lung disease.

Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains

BackgroundEscherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overflow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to find the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two different approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation.ResultsWe evaluated protein overexpression in E. coli BL21 wt and five mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efficient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fivefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation.ConclusionThe results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fluxes to different pathways and affect the final yield of the recombinant protein bioprocess. Thereby, we obtained a fivefold increase in protein production and an efficient use of the resources employing the most suitable strain and culture conditions.

Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis

ABSTRACTAcetyl phosphate (AcP) is generally produced from acetyl coenzyme A by phosphotransacetylase (Pta), and subsequent reaction with ADP, catalyzed by acetate kinase (Ack), produces ATP. The mechanism of ATP production in Porphyromonas gingivalis is poorly understood. The aim of this study was to explore the molecular basis of the Pta-Ack pathway in this microorganism. Pta and Ack from P. gingivalis ATCC 33277 were enzymatically and structurally characterized. Structural and mutational analyses suggest that Pta is a dimer with two substrate-binding sites in each subunit. Ack is also dimeric, with a catalytic cleft in each subunit, and structural analysis indicates a dramatic domain motion that opens and closes the cleft during catalysis. ATP formation by Ack proceeds via a sequential mechanism. Reverse transcription-PCR analysis demonstrated that the pta (PGN_1179) and ack (PGN_1178) genes, tandemly located in the genome, are cotranscribed as an operon. Inactivation of pta or ack in P. gingivalis by homologous recombination was successful only when the inactivated gene was expressed in trans. Therefore, both pta and ack genes are essential for this microorganism. Insights into the Pta-Ack pathway reported herein would be helpful to understand the energy acquisition in P. gingivalis.

Impact of poxB, pta, and ackA genes on mineral-weathering of Enterobacter cloacae S71.

In this study, biotite weathering behaviors were compared between mineral-weathering bacteria Enterobacter cloacae S71, mutant strains created by the deletion of poxB, pta, and ackA genes involved in acetate formation, and their complemented strains. Compared to strain S71, a decrease in bacterial growth was observed during the early and middle stages for the mutant ΔpoxB and at the middle and later stages for the mutants Δpta and ΔackA. Dissolved Al and Fe concentrations were lower during the early stage for strain ΔpoxB, at the early or middle stage for strain Δpta, and at the middle and later stages and throughout the weathering process for strain ΔackA, compared to strain S71. Acetate production was depressed during the early stage for strain ΔpoxB, at the early and middle stages for strain Δpta, and throughout the weathering process for strain ΔackA. Overall, the ackA gene exhibited a larger impact on dissolved Fe and acetate concentrations than both the poxB and pta genes. Reduced bacterial growth and lower dissolved Al, Fe, and acetate concentrations recovered by the complemented strains. These results show that strain S71 promoted mineral weathering through the production of acetic acid with distinctive impacts by the genes involved in acetate.

Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens.

There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. 13 C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using 13 C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.

Metabolic and stress responses of Acinetobacter oleivorans DR1 during long-chain alkane degradation.

Summary Acinetobacter oleivorans DR1 can utilize C12–C30 alkanes as a sole carbon source but not short‐chain alkanes (C6, C10). Two copies of each alkB‐, almA‐ and ladA‐type alkane hydroxylase (AH) are present in the genome of DR1 cells. Expression and mutational analyses of AHs showed that alkB1 and alkB2 are the major AH‐encoding genes under C12–C30, and the roles of other almA‐ and ladA genes are negligible. Our data suggested that AlkB1 is responsible for long‐chain alkane utilization (C24–C26), and AlkB2 is important for medium‐chain alkane (C12–C16) metabolism. Phylogenetic analyses revealed large incongruities between phylogenies of 16S rRNA and each AH gene, which implies that A. oleivorans DR1 has acquired multiple alkane hydroxylases through horizontal gene transfer. Transcriptomic and qRT‐PCR analyses suggested that genes participating in the synthesis of siderophore, trehalose and poly 3‐hydroxybutyrate (PHB) were expressed at much higher levels when cells used C30 than when used succinate as a carbon source. The following biochemical assays supported our gene expression analyses: (i) quantification of siderophore, (ii) measurement of trehalose and (iii) observation of PHB storage. Interestingly, highly induced both ackA gene encoding an acetate kinase A and pta gene encoding a phosphotransacetylase suggested unusual ATP synthesis during C30 alkane degradation, which was demonstrated by ATP measurement using the ΔackA mutant. Impaired growth of the ΔaceA mutant indicated that the glyoxylate shunt pathway is important when C30 alkane is utilized. Our data provide insight into long‐chain alkane degradation in soil microorganisms.

CcpA and CodY Coordinate Acetate Metabolism in Streptococcus mutans.

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) and acetate kinase (Ack) convert pyruvate into acetate with the concomitant generation of ATP. The genes for this pathway are tightly regulated by multiple environmental and intracellular inputs, but the basis for differential expression of the genes for Pta and Ack in S. mutans had not been investigated. Here, we show that inactivation in S. mutans of ccpA or codY reduced the activity of the ackA promoter, whereas a ccpA mutant displayed elevated pta promoter activity. The interactions of CcpA with the promoter regions of both genes were observed using electrophoretic mobility shift and DNase protection assays. CodY bound to the ackA promoter region but only in the presence of branched-chain amino acids (BCAAs). DNase footprinting revealed that the upstream region of both genes contains two catabolite-responsive elements (cre1 and cre2) that can be bound by CcpA. Notably, the cre2 site of ackA overlaps with a CodY-binding site. The CcpA- and CodY-binding sites in the promoter region of both genes were further defined by site-directed mutagenesis. Some differences between the reported consensus CodY binding site and the region protected by S. mutans CodY were noted. Transcription of the pta and ackA genes in the ccpA mutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription of ackA and pta in S. mutans that optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH.IMPORTANCE The human dental caries pathogen Streptococcus mutans is remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway in S. mutans modulates carbohydrate flux and fine-tunes the ability of the organisms to cope with stressors that are commonly encountered in the oral cavity. Here, we show that CcpA controls transcription of the pta and ackA genes via direct interaction with the promoter regions of both genes and that branched-chain amino acids (BCAAs), particularly isoleucine, enhance the ability of CodY to bind to the promoter region of the ackA gene. A working model is proposed to explain how regulation of pta and ackA genes by these allosterically controlled regulatory proteins facilitates proper carbon flow and energy production, which are essential functions during infection and pathogenesis as carbohydrate and amino acid availability continually fluctuate.

Amino Acid Catabolism in Staphylococcus aureus and the Function of Carbon Catabolite Repression.

ABSTRACTStaphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kinase, also abrogated growth of JE2 in CDM, suggesting that ATP production from pyruvate-producing amino acids is also critical for growth. In addition, although a functional respiratory chain was absolutely required for growth, the oxygen consumption rate and intracellular ATP concentration were significantly lower during growth in CDM than during growth in glucose-containing media. Finally, transcriptional analyses demonstrated that expression levels of genes coding for the enzymes that synthesize glutamate from proline, arginine, and histidine are repressed by CcpA and carbon catabolite repression. These data show that pathways important for glutamate catabolism or ATP generation via Pta/AckA are important for growth in niches where glucose is not abundant, such as abscesses within skin and soft tissue infections.

Deletion of poxB, pta, and ackA improves 1,3-propanediol production by Klebsiella pneumoniae.

To date, few studies have focused on reducing the toxic by-product acetate during 1,3-propanediol production by Klebsiella pneumoniae. In this study, the effects of deleting the poxB, pta, and ackA genes, which are involved in the two main acetate synthesis pathways, on cell growth and 1,3-propanediol production were investigated. Although acetate synthesis via pyruvate oxidase (PoxB, encoded by poxB) generally seems unnecessary and wasteful, PoxB was shown to play an important role in K. pneumoniae. Deletion of poxB severely inhibited cell growth, and the poxB mutant exhibited an anomalously high accumulation of acetate in aerobic cultures and failed to produce an endogenous supply of carbon dioxide (CO2) in anaerobic cultures. It is interesting that both the aerobic and anaerobic growth defects of the poxB mutant were corrected by further deleting pta and ackA, which blocked the other main acetate synthesis pathway. The poxB-pta-ackA mutant excreted less acetate and showed an excellent ability to produce 1,3-propandiol. The final 1,3-propanediol yield and concentration in a 2-L fed-batch fermentation reached 0.66 (mol/mol) and 76.8 g/L, respectively, which were 16 and 15 % greater, respectively, than those of the parent strain.