Siegfried M. Schoberth

NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum.

ABSTRACT Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with 31P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.

Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum

The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain length; the protein was most active with PolyP75. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4% (w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions of glucose excess, the presence of PPGK entailed a growth advantage.

Desulfovibrio furfuralis sp. nov., a furfural degrading strictly anaerobic bacterium

Summary Physiological, biochemical and taxonomic characteristics of the strictly anaerobic sulfate reducing bacterium Desulfovibrio sp. strain F-1 have been studied. Optimal growth on furfural as sole source of carbon occurred at 37°C and pH 6.8. Furfural, furfurylalcohol and 2-furoic acid were fermented to acetate with the concurrent reduction of sulfate to sulfide. Sulfite or nitrate could replace sulfate as electron acceptor. During metabolism of (carbonyl- 13 C)-furfural, 2- 13 C-furfurylalcohol and 2- 13 C-furoic acid could be detected.as intermediates and the 13 C-labelled carbonyl-group was converted to 13 CO 2 ; this indicates intermediary decarboxylation of the heteraromatic compound. On the basis of oligonucleotide cataloguing Desulfovibrio sp. strain F1 is suggested to be a new species, Desulfovibrio furfuralis . It had a DNA base composition of 61 mol % guanine plus cytosine.

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.

Exopolyphosphatases PPX1 and PPX2 from Corynebacterium glutamicum.

ABSTRACT Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (ppx1) and cg1115 (ppx2) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Δppx1 and Δppx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum. PPX2His was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg2+ or Mn2+ cations but was inhibited by millimolar concentrations of Mg2+, Mn2+, and Ca2+. PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.

Monitoring of inorganic polyphosphate dynamics in Corynebacterium glutamicum using a novel oxygen sparger for real time 31P in vivo NMR

For the first time in intact bacterial cells, the dynamics of the build-up of soluble cytosolic inorganic polyphosphate (polyP) during aeration, and its breakdown during anaerobiosis have been observed with a time resolution of 50 s. Under conditions of 60-80% saturation with pure oxygen, the accumulation of high levels of intracellular polyP was detected when inorganic phosphate (Pi) and glucose or acetate were added to Corynebacterium glutamicum cell suspensions (3 ml, ∼40 mg dw/ml). The maximum levels of polyP reached were estimated to 600 mM P units in the cytosol or ∼3% phosphorus [w/w] in the cell dry weight. C. glutamicum polyP was apparently of high molecular weight (containing probably a few hundred units) as inferred from signal distribution, but a temporary average polyP chain length of about n = 40 could be estimated at the initial stages of polyP formation. After each addition of glucose or acetate, oxygen levels followed a steep decline to ∼20% and then an increase to the previous level. In contrast, polyP levels rose after the addition of substrate, and declined again, while the oxygen level recovered. When the oxygen supply was completely switched off, the polyP signal declined immediately, with concomitant re-appearance of phosphomonoester signals (sugar phosphates and related compounds). Both processes, the increase of polyP during aeration and supply with substrate and Pi, and the decrease during anaerobiosis, occurred within minutes. Only within these relatively brief windows of time between successive feedings with substrate or between aeration and anaerobiosis, high levels of polyP could be observed. Thus, our findings indicate that polyP occurs not only as the long known granular storage material in some Corynebacteria, such as C. diphtheriae or C. imitans, but that formation and breakdown of soluble polyP in C. glutamicum is a very dynamic process that may play a decisive role in C. glutamicum and in other strains of this genus. These investigations were made possible by combining nuclear magnetic resonance (NMR) techniques with novel methods of oxygen sparging and online substrate distribution. The sparger was custom made from titanium to fit into 10 mm o.d. NMR tubes. Both the size and the spacing of the holes in the sparger were calculated for optimum distribution of oxygen at 30 °C through 3 ml of C. glutamicum cell suspensions. The experiments were carried out using in vivo31P NMR, and monitoring of oxygen was performed with a miniature oxygen optode in real time. Glucose or acetate and/or phosphate stock solutions could be added in situ.31P NMR analyses of intracellular phosphorus metabolites were sampled with a time resolution of 50 s. The sparger unit, including optode and supply lines, could be easily switched from one sample to another after completion of an experiment. It is suggested to use these analytical tools to investigate other bacterial strains and even cell extracts, shedding further light on the novel roles of polyP in living cells [Schroder, H. C., Muller, W. E. G., (eds.). Inorganic polyphosphates - biochemistry, biology, biotechnology. Prog. Mol. Subcell. Biol. 23 (1999). Springer-Verlag, Berlin].

Production of acetic acid by Acetogenium kivui

SummaryThe formation of acetic acid by the thermophilic nonsporeforming homoacetogenic bacterium Acetogenium kivui was studied under various conditions. In pH-controlled batch fermentation at pH 6.4 this bacterium was able to produce up to 625 mM of acetic acid from glucose within 50–60 h. The value of μmax obtained was about 0.17 h-1, the yield was about 2.55 mol of acetic acid per mol of glucose utilized. In continuous fermentation both substrate concentration and dilution rate (D) influenced the yield of acetate and the stationary concentration: a glucose concentration of 67 mM at D=0.09 h-1 resulted in 2.82 mol acetate/mol glucose and 190 mM acetate at a production rate of 17.1 mM/1 h. When the dilution rate was increased the production rate reached a maximal value of 43.2 mM/1 h at D=0.32 h-1. At a glucose concentration of 195 mM the dependence of yield upon dilution rate followed a similar pattern and an acetate concentration of 420 mM could be obtained. Enzymatic studies indicate that in A. kivui pyruvate ferredoxin-oxidoreductase and acetate kinase are inhibited at acetate concentrations higher than 800 mM. Based on these results a fed-batch fermentation was developed, which allowed to produce more than 700 mM acetic acid within 40–50 h.

Anaerobic degradation of sulphite evaporator condensate in a fixed-bed loop reactor by a defined bacterial consortium

SummaryAfter elucidating the composition of an anaerobic bacterial enrichment culture treating sulphite evaporator condensate (SEC), an effluent in the pulp and paper industry, we built up stepwise a defined mixed culture to convert the organic constituents of SEC (acetate, methanol, furfural) to methane and CO2. In batch cultures Desulfovibrio furfuralis and Methanobacterium bryantii degraded furfural in the absence of sulphate via inter-species H2 transfer yielding 0.42 mol methane and 1.87 mol acetate/mol furfural degraded. When Methanosarcina barkeri was added to this diculture, acetate was also transformed to methane yielding 0.93 mol methane/mol acetate converted. This consortium (D. furfuralis, Methanobacterium bryantii and Methanosarcina barkeri) degraded furfural in continuous culture (fixed-bed loop reactor) to 92%, but the conversion of acetate was only 67%. The conversion of acetate could be further improved to 86% by adding 10 mm sulphate to the medium. This resulted in a space time yield of 10.9 g chemical oxygen demand (COD)/1 per day for the overall conversion. With a consortium consisting of M. barkeri, Methanobrevibacter arboriphilus, Methanosaeta concilii and D. furfuralis, a synthetic SEC could be degraded at a space time yield of 13.35 g COD/1 per day. This defined culture degraded all the constituents of SEC at an efficiency of almost 90% compared to an enrichment culture under identical conditions.