Coordinated expression of acetyl CoA synthetase and the ace operon enzymes in Escherichia coli in preparation for adaptation to acetate.

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.

High-titre production of aromatic amines in metabolically engineered Escherichia coli.

AIMS: Aromatic amines with diverse physical characteristics are often employed as antioxidants and precursors to pharmaceutical products. As the traditional chemical methods pose serious environmental pollution, there is an arising interest in biomanufacturing aromatic amines from renewable feedstocks. MATERIALS AND RESULTS: We report the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine and phenylethylamine. First, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with increasing shikimate (SHK) pathway flux towards L-tyrosine. We found that glycerol served as a better carbon source than glucose, resulting in 940 ± 46 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), phosphate acetyltransferase (pta) and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation by-product formation. The tyramine level was further increased to 1.965 ± 0.205 g/L in the shake flask, which was improved by 2.1 times compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 ± 21 mg/L dopamine and 555 ± 50 mg/L phenethylamine. CONCLUSIONS: We demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy for improving aromatic amine productions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study achieved the highest aromatic amine titres in E. coli under shake flask reported to date.

Cognate sensor kinase-independent activation of Mycobacterium tuberculosis response regulator DevR (DosR) by acetyl phosphate: implications in anti-mycobacterial drug design.

The DevRS/DosT two-component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under 'non-inducing' aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase-phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate-grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild-type DevS and phosphorylation-defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous-like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate-dependent phosphorylation can occur in the absence of DevR's cognate kinases. Based on our findings, we conclude that anti-mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.

The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.

Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum.

BACKGROUND: Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. RESULTS: In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively. CONCLUSION: Our results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi).

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem. We then applied CRISPRi in a strain engineered for 3-hydroxybutyrate (3HB) production to examine targets for increasing carbon flux toward the desired product. Downregulating phosphotransacetylase (pta) with a single sgRNA led to a 97% decrease in enzyme activity and a 2.3-fold increase in titer during heterotrophic growth. However, acetate production still accounted for 40% of the carbon flux. Repression of aldehyde:ferredoxin oxidoreductase (aor2), another potential route for acetate production, led to a 5% reduction in acetate flux, whereas using an additional sgRNA targeted to pta reduced the enzyme activity to 0.7% of the wild-type level, and further reduced acetate production to 25% of the carbon flux with an accompanying increase in 3HB titer and yield. These results demonstrate the utility of CRISPRi for elucidating and controlling carbon flow in C. ljungdahlii.

Rational design and analysis of an Escherichia coli strain for high-efficiency tryptophan production.

L-tryptophan (L-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient L-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the L-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG fbr ). To further improve the production of L-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of L-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.

Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis.

Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes.

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.

Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum.

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg-1) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073.

For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.

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.

Alternative acetate production pathways in Chlamydomonas reinhardtii during dark anoxia and the dominant role of chloroplasts in fermentative acetate production.

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Decreased formation of branched-chain short fatty acids in Bacillus amyloliquefaciens by metabolic engineering.

OBJECTIVES: To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared. RESULTS: By knockout of the ptb gene, 1.01 g BCFAs kg-1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg-1). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg-1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12. CONCLUSIONS: HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

Insights from the complete genome sequence of Clostridium tyrobutyricum provide a platform for biotechnological and industrial applications.

Genetic research enables the evolution of novel biochemical reactions for the production of valuable chemicals from environmentally-friendly raw materials. However, the choice of appropriate microorganisms to support these reactions, which must have strong robustness and be capable of a significant product output, is a major difficulty. In the present study, the complete genome of the Clostridium tyrobutyricum strain CCTCC W428, a hydrogen- and butyric acid-producing bacterium with increased oxidative tolerance was analyzed. A total length of 3,011,209 bp of the C. tyrobutyricum genome with a GC content of 31.04% was assembled, and 3038 genes were discovered. Furthermore, a comparative clustering of proteins from C. tyrobutyricum CCTCC W428, C. acetobutylicum ATCC 824, and C. butyricum KNU-L09 was conducted. The results of genomic analysis indicate that butyric acid is produced by CCTCC W428 from butyryl-CoA through acetate reassimilation via CoA transferase, instead of the well-established phosphotransbutyrylase-butyrate kinase pathway. In addition, we identified ten proteins putatively involved in hydrogen production and 21 proteins associated with CRISPR systems, together with 358 ORFs related to ABC transporters and transcriptional regulators. Enzymes, such as oxidoreductases, HNH endonucleases, and catalase, were also found in this species. The genome sequence illustrates that C. tyrobutyricum has several desirable traits, and is expected to be suitable as a platform for the high-level production of bulk chemicals as well as bioenergy.

Two propanediol utilization-like proteins of Moorella thermoacetica with phosphotransacetylase activity.

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood-Ljungdahl (acetyl-CoA) pathway in which CO2 is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood-Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class(III)-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).

Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum.

Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and Pi. Two classes of Pta have been identified based on the absence (Pta(I)) or presence (Pta(II)) of an N-terminal regulatory domain. Pta(I) has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica Pta(II) enzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycete Phytophthora ramorum. The two Ptas from P. ramorum, designated PrPta(II)1 and PrPta(II)2, both belong to class II. PrPta(II)1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPta(II)1 and the S. enterica Pta(II) and found that, although the N-terminal regulatory domains share only 19% identity, both enzymes are inhibited by ATP, NADP, NADH, phosphoenolpyruvate (PEP), and pyruvate in the acetyl-CoA/Pi-forming direction but are differentially regulated by AMP. Phylogenetic analysis of bacterial, archaeal, and eukaryotic sequences identified four subtypes of Pta(II) based on the presence or absence of the P-loop and DRTGG subdomains within the N-terminal regulatory domain. Although the E. coli, S. enterica, and P. ramorum enzymes all belong to the IIa subclass, our kinetic analysis has indicated that enzymes within a subclass can still display differences in their allosteric regulation.

The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment.

UNLABELLED: In Salmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology. IMPORTANCE: Bacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous proteins to the lumen of the Pdu MCP. These findings expand our general understanding of bacterial MCP assembly and cofactor homeostasis.

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans.

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.

Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens.

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas.