Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism.

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (Delta malE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.

Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation.

To give clues about the respective importance of phosphoenol-pyruvate carboxylase (PEPc) and pyruvate carboxylase (Pc) in Corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, PEPc activity was genetically amplified and Pc activity was suppressed by biotin limitation in the culture medium. In absence of Pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. Whereas PEPc amplification in excess of biotin (4 mg/L) only slightly modified the cell kinetics, under biotin limiting conditions this amplification strongly improved the glutamate production (4 microg/L). When Pc was absent, PEPc activity was sufficient to allow up to 70% of the maximal glutamate production rate and seemed to have an important anaplerotic role, especially at the beginning of the production phase. In contrast, Pc was predominant during the remainder of the glutamate fermentation.

A protein related to eucaryal and bacterial DNA-motor proteins in the hyperthermophilic archaeon Sulfolobus acidocaldarius.

We have isolated a new gene encoding a putative 103-kDa protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Analysis of the deduced amino-acid sequence shows an extended central domain, predicted to form coiled-coil structures, and two terminal domains that display purine NTPase motifs. These features are reminiscent of mechanochemical motor proteins which use the energy of ATP hydrolysis to move specific cellular components. Comparative analysis of the amino-acid sequence of the terminal domains and predicted structural organization of this putative purine NTPase show that it is related both to eucaryal proteins from the "SMC family" involved in the condensation of chromosomes and to several bacterial and eucaryal proteins involved in DNA recombination/repair. Further analyses revealed that these proteins are all members of the so called "UvrA-related NTP-binding proteins superfamily" and form a large subgroup of motor-like NTPases involved in different DNA processing mechanisms. The presence of such protein in Archaea, Bacteria, and Eucarya suggests an early origin of DNA-motor proteins that could have emerged and diversified by domain shuffling.

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile.

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.

A common organization of the T-DNA genes expressed in plant hairy roots induced by different plasmids of Agrobacterium rhizogenes.

The recently described pathogenic plasmid from Agrobacterium rhizogenes 2659 induces "hairy root" proliferation in infected plants which synthesize a new opine type, provisionally called cucumopine. The T-DNA restriction site map of 2659 differs from other pRi T-DNAs, i.e., mannopine and agropine. However all these three different T-DNAs have a similar organization as detected through hybridization. This, previously shown by our cross-hybridization studies between mannopine and agropine pRi T-DNAs, is here extended to the cucumopine pRi T-DNA; each of the three pRi T-DNA is composed of successive DNA regions that would find actual counterparts in the two other T-DNAs. Equivalence of DNA regions is suggested by their similar length, location and organization in the three T-DNAs and by their sequence homology substantial enough to be reproducibly cross-hybridized in all our experiments. This suggests that the three pRi T-DNAs share most of their functions. This was sought by analysing the pRi 2659 T-DNA gene expression in transformed plant tissues. The mRNA species observed in pRi 2659 carrot hairy roots of in vitro cultures give a pattern that is rather unvariable and resembles those described for some agropine type pRi transformant tissues. This transcript pattern is consistent with the preservation in the pRi 2659 T-DNA of counterparts of most of the open reading frames detected in the TL-DNA of agropine type pRis.