The Arnon-Buchanan cycle: a retrospective, 1966-2016.

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO2, reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO2. Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

Multiple-locus variable-number tandem repeat analysis of Legionella pneumophila using multi-colored capillary electrophoresis.

Several methods for typing of Legionella pneumophila exist, one of which is an 8-locus variable-number of tandem repeats analysis (MLVA). This method is based on separating and sizing amplified VNTR PCR products by agarose gel electrophoresis. In the present work, the existing L. pneumophila MLVA-8 assay is adapted to capillary electrophoresis. The assay was multiplexed by using multiple fluorescent labeling dyes and tested on a panel of L. pneumophila strains with known genotypes. The results from the capillary electrophoresis-based assay are shown to be equivalent to, and in a few cases more sensitive than, the gel-based genotyping assay. The assay presented here allows for a swift, automated and precise typing of L. pneumophila from patient or environmental samples and represents an improvement over the current gel-based method.

Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx1 and stx2) and the Intimin gene (eae).

OBJECTIVES: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coli outer membrane protein (Eae) or intimin, encoded by the stx and eae genes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stx and eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. RESULTS: The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic.

Parallel nanoliter detection of cancer markers using polymer microchips.

A general multipurpose microchip technology platform for point-of-care diagnostics has been developed. Real-time nucleic acid sequence-based amplification (NASBA) for detection of artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples was successfully performed in cyclic olefin copolymer (COC) microchips, incorporating supply channels and parallel reaction channels. Samples were distributed into 10 parallel reaction channels, and signals were simultaneously detected in 80 nl volumes. With a custom-made optical detection unit, the system reached a sensitivity limit of 10(-6) microM for artificial HPV 16 sequences, and 20 cells microl(-1) for the SiHa cell line. This is comparable to the detection limit of conventional readers, and clinical testing of biological samples in polymer microchips using NASBA is therefore possible.

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface.

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.

Electrostatic interactions across the dimer-dimer interface contribute to the pH-dependent stability of a tetrameric malate dehydrogenase.

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic bacteria usually are dimers. Using site-directed mutagenesis, we show here that a network of electrostatic interactions across the extra dimer-dimer interface in CaMDH is important for thermal stability and oligomeric integrity. Stability effects of single point mutations (E25Q, E25K, D56N, D56K) varied from -1.2 degrees C to -26.8 degrees C, and depended strongly on pH. Gel-filtration experiments indicated that the 26.8 degrees C loss in stability observed for the D56K mutant at low pH was accompanied by a shift towards a lower oligomerization state.

Rational engineering of enzyme stability.

During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.

Real-time nucleic acid sequence-based amplification in nanoliter volumes.

Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.