Prosorhynchine trematodes (Digenea: Bucephalidae) from epinephelines (Perciformes: Serranidae) on the Great Barrier Reef,Australia

Six new species of bucephalid trematodes from the prosorhynchine bucephalid genera Prosorhynchus Odhner, 1905 and Neidhartia Nagaty, 1937 are reported from the epinepheline genera Cephalopholis, Cromileptes, Epinephelus and Variola on the Great Barrier Reef, Australia. Two species of Prosorhynchus and one of Neidhartia are reported from Epinephelus spp., P. jexi n. sp. from E. quoyanus, P. lafii n. sp. from E. fuscoguttatus and N. epinepheli n. sp. from E. maculatus. The other three new species are P. robertsthomsoni n. sp. from Cephalopholis argus, C. cyanostigma and C. miniata, P. conorjonesi n. sp. from Cromileptes altivelis, and P. milleri n. sp. from Variola louti. Extensive examinations of other piscivorous fish species from the Great Barrier Reef have not revealed these six bucephalid species, which appear to be restricted at least to the host genera from which they are reported here.     

Link between Phosphate Starvation and Glycogen Metabolism in Corynebacterium glutamicum,Revealed by Metabolomics

In this study, we analyzed the influence of phosphate (P_i) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under P_i limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under P_i excess, the glycogen level in P_i-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under P_i excess, did so under P_i limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under P_i limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited P_i concentrations.Phosphorus is an essential nutrient for all cells and is required for, e.g., the biosynthesis of nucleotides, NAD(P)H, DNA, and RNA but also for the regulation of protein activity by phosphorylation of histidine, aspartate, serine, threonine, or tyrosine residues. A common phosphorus source is inorganic phosphate (P_i), and cells have developed mechanisms for the acquisition, assimilation, and storage of P_i. When P_i becomes limiting, many bacteria induce the synthesis of proteins that enable them to capture the residual P_i resources more efficiently and to make alternative phosphorus sources accessible. The corresponding genes are collectively named P_i starvation-inducible genes, or psi genes. The P_i starvation response, and in particular its regulation, has been most carefully studied in Escherichia coli (45) and Bacillus subtilis (14).We recently started to characterize the P_i starvation response in Corynebacterium glutamicum, a Gram-positive soil bacterium used industrially for the production of more than two millions tons of amino acids per year, mainly l-glutamate and l-lysine (12). An overview of the biology, genetics, physiology, and application of C. glutamicum can be found in two recent monographs (3, 6). Phosphorus constitutes 1.5% to 2.1% of the cell dry weight of C. glutamicum (24), part of which can be present as polyphosphate (22, 29). Several of the enzymes involved in polyphosphate metabolism have been characterized recently, such as a class II polyphosphate kinase (28), the exopolyphosphatases Ppx1 and Ppx2 (26), a polyphosphate/ATP-dependent glucokinase (25), and a polyphosphate/ATP-dependent NAD⁺ kinase (27). The P_i starvation stimulon of C. glutamicum was determined using whole-genome DNA microarrays (15). Comparison of the mRNA profiles before and at different times after a shift from P_i excess to P_i starvation led to the identification of a group of genes that are presumably required to cope with limited P_i supply. This group includes the following: the pstSCAB operon, encoding an ABC transporter for high-affinity P_i uptake; the ugpAEBC operon, encoding an ABC transporter for uptake of glycerol 3-phosphate; glpQ1, encoding a glycerophosphoryl diester phosphodiesterase; ushA, encoding a secreted enzyme with UDP-sugar hydrolase and 5′-nucleotidase activities (33); nucH, encoding a putative secreted nuclease which possibly plays a role in liberating P_i from extracellular nucleic acids; phoC (NCgl2959/cg3393), which may encode a cell wall-associated phosphatase (46); phoH1, encoding an ATPase of unknown function; and the pctABCD operon, encoding an ABC transport system which might be involved in the uptake of a yet-unknown phosphorus-containing compound (15). C. glutamicum lacks homologs of genes for phosphonate degradation, as well as the capability to utilize phosphonates as P sources (15).In most bacteria analyzed in this respect, the P_i starvation response is controlled by two-component signal transduction systems, e.g., the PhoBR system in E. coli (13) and the PhoPR system in B. subtilis (14). Our previous studies revealed that in C. glutamicum, a two-component system composed of the sensor kinase PhoS and the response regulator PhoR is involved in the activation of phosphate starvation-inducible genes (21). Studies with purified proteins showed that phosphorylation by PhoS increased the DNA-binding affinity of PhoR, which bound to many of the P_i starvation-inducible genes, but with different affinities (34).The study reported here was initiated by the question how the metabolism of C. glutamicum responds to P_i limitation. Our results reveal a link between P_i limitation and glycogen metabolism, which was also used for metabolic simulations based on a genome-wide metabolic model.     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids

Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine.     

Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine

Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 DeltailvA DeltapanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.     

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.     

Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA

The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compounds. We recently showed that the separated periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a K(d) of 5.5 microM at pH 7. To identify positively charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titration calorimetry (ITC). In 12 cases, the K(d) for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 microM). In one case (R98A), the K(d) was 6-fold decreased (0.8 microM), whereas in four cases (R66A, H69A, R107A, and K109A) the K(d) was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV circular dichroism (CD) spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the melting point (T(m)) of wild-type CitAP and mutein R98A by 6.2 and 9.5 degrees C, respectively, but had no effect on the T(m) of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.     

The Escherichia coli Citrate Carrier CitT: a Member of a Novel Eubacterial Transporter Family Related to the 2-Oxoglutarate/Malate Translocator from Spinach Chloroplasts

Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol, Escherichia coli converts citrate to acetate and succinate. Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase. Here we report that the open reading frame (designated citT) located at 13.90 min on the E. coli chromosome between rna and the citrate lyase genes encodes a citrate carrier. E. coli transformed with a plasmid expressing citT was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier. Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate. Since succinate is the end product of citrate fermentation in E. coli, it is likely that CitT functions in vivo as a citrate/succinate antiporter. Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices. Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized. It is suggested that the E. coli CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.     

Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis.

We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type. Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane. Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3.