William Chesbro

Eubacteria have 3 growth modes keyed to nutrient flow

Aerobic growth of Escherichia coli and Paracoccus denitrificans has been studied in chemostat, fed batch, and recycling fermentor modes under carbon and energy limitation. Two abrupt drops or discontinuities in molar growth yield, Y, have been found that occur over relatively short ranges in the value of specific growth rate.Before the first discontinuity, Y is constant and maximal. After the first discontinuity, at a doubling time of 33 h, Y becomes constant again and independent of μ until the second discontinuity appears at a doubling time of about 50 h, corresponding to a μ of about 0.014. At this point, Y drops to a lower value that is constant at doubling times longer than 100 h, corresponding to a μ of about 0.007.The second discontinuity is associated in Paracoccus with elevated levels of guanosine tetraphosphate (ppGpp) that impose stringent regulation as has been found previously with Bacillus and Escherichia species. It is thus likely that the stringent response generally occurs in bacteria in vivo at a doubling time of about 50 h. The cause of the first discontinuity is unknown. All experiments indicate that Pirt-type calculations relating μ, Y, and maintenance energy demand are no longer valid. In chemostat experiments, the intercept of the relationship between specific substrate utilization and specific growth rate is defined as maintenance. However, this intercept most probably is caused by stringent regulation at low dilution rates. Three regions of bacterial growth rates are defined by this study, corresponding to doubling times of 0.5 to 15 h, 33 to 50 h, and >100 h. Some growth behavior in each region is unique to that region.

Electro-transformation of Clostridium beijerinckii NRRL B-592 with shuttle plasmid pHR106 and recombinant derivatives

Conditions for transformation of the solventogenic anaerobe Clostridium beijerinckii NRRL B-592 with plasmid DNA via electroporation are described. Shuttle plasmid pHR106 and two derivatives constructed in this study were transferred and were expressed in this organism. One recombinant derivative of pHR106 was constructed by separately subcloning the clostridial tetracycline (tetP) resistance genes into pHR106. The second vector conferring erythromycin resistance was obtained via in-vivo recombination. The new constructs, termed pRZL and pRZE respectively, were then transferred to C. beijerinckii in order to evaluate their potential as shuttle vectors. The recombinant plasmids were shown to transfer to C. beijerinckii and were expressed as autonomously replicating vectors. The use of these plasmids as cloning and shuttle vectors for C. beijerinckii is discussed.

Continuous culture of bacteria with biomass retention

Abstract Bacterial cells have three phases of growth which are characterized respectively by: (1) balanced growth with a high yield of biomass; (2) balanced growth with lower biomass yield; and (3) unbalanced growth with lowest biomass yield. Phases 2 and 3 are associated with elevated concentrations of the regulatory nucleotides centered on guanosine-5′-diphosphate-3′-diphosphate. Maintenance of the correct growth phase is important in optimizing industrial product formation by bacterial populations.

relA and Related Loci are Growth Rate Determinants for Escherichia coli in a Recycling Fermenter

SUMMARY: Three sharply distinct phases were found in the growth of Escherichia coli in a continuous fermenter with 100% cell recycle. They were: an initial phase of batch-type growth reaching exponential rates that terminated abruptly when the glucose concentration in the fermenter fell below the cells transport threshold; a second phase of linear growth whose rate was dependent on the rate of glucose provision to the fermenter, but whose length was fixed and independent of growth rate; and a third phase of slower linear growth, whose rate was also dependent on the glucose provision rate, characterized by a restriction in RNA accumulation, and indefinite in length. Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) accumulation commenced at the start of phase 2 and rose throughout the phase. It reached a maximum at the start of phase 3, concurrent with the curbing of RNA accumulation; thereafter, the ppGpp concentration was constant unless the rate of glucose supply was increased, at which point it fell to a phase 2 level and the cells returned to a corresponding phase 2 growth rate. The length of growth phase 2 was changed in a precise manner by mutations in the relA, relX and spoT loci. Phase 2 was longer for relA and relX mutants (decreased capability for ppGpp synthesis) and shorter for spoT mutants (decreased capability for ppGpp degradation). It is concluded that the continuously falling energy supply per cell in a recycling fermenter invokes ppGpp accumulation by at least three biochemical mechanisms and that the third, final growth phase in the fermenter is an indefinitely extended manifestation of strong regulation by ppGpp.

Growth rate dependence of solventogenesis and solvents produced byClostridium beijerinckii

SummaryAcidogenesis and solventogenesis byClostridium beijerinckii NRRL B-593 has been studied in batch growth, and in sucrose-limited chemostat and recycling fermentor growth. Cells grown in batch culture without pH control primarily produced either butyric and acetic acids, or these acids plus butanol, ethanol and isopropanol in ratios depending on the mediums content of reducing agent, calcium and iron. Cells in chemostat-culture at a mass doubling time (td) of 5.8 h produced primarily butyric and acetic acids at pH 6.8 and these acids plus butanol, ethanol and isopropanol at pH 4.8. Cells grown in a recycling fermentor (in which the td continuously increases) at pH 6.8 entered solventogenesis at a td of 43 h, producing primarily propanol, ethanol and butanol, along with butyric acid, but with greatly decreased production of acetic acid. Although “clostridial form” morphology, succeeded by sporulation, usually accompanied solventogenesis, the association was not invariant so that solventogenesis and sporogenesis can occur separately in this species.

Maintenance energy demand affects biomass synthesis but not cellulase production by a mesophilicClostridium

SummarySecretion of cellulolytic activity by the mesophilClostridium strain C7 was studied while the bacterium underwent progressive carbon/energy starvation and the ensuing continuous decline in growth rate. In the slowest range of growth rates studied the organism was in full response to the global regulation imposed by guanosine 5′, 3′-bispyrophosphate (ppGpp). The exoenzymes of the cellulase complex were produced at the same volumetric rate whether or not the response was active. However, the volumetric rate of biomass synthesis was reduced 45% or more by the response. Energy necessary to maintain the ppGpp-regulated state (i.e., maintenance energy) was, therefore, diverted from energy going to synthesis of biomass but not from that going to exoenzyme synthesis, making the yield of cellulase activity per mole of carbon-energy substrate independent of growth rate and the exoenzyme complex produced from the substrate with equal efficiency at all growth rates. The primary consideration in improving exoenzyme productivity by bacteria with this type of energy distribution between secretion, growth, and maintenance is simply increasing yield per mole of carbon-energy substrate, with growth rate effects on yield a secondary and minimum concern.

Protoplast formation, L-colony growth, and regeneration ofClostridium beijerinckii NRRL B-592 and B-593 andClostridium acetobutylicum ATCC 10132

SummaryProtocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques.

POOLS, CONFLUXES AND TRANSPORT OF AMINO ACIDS IN STREPTOCOCCUS FAECIUM.

Abstract 1. 1. In OH−-dependent, reversed amino acid transport in Streptococcus faecium it is possible to distinguish the involvement of four confluxes, i.e., groups of amino acids which are released in synchrony from the cell by either a tert.-butanol-isopropanol mixture, trichloroacetic acid, spermine, NH2OH, or OH−. 2. 2. Conflux 1 (released by the solvent mixture) represents the cells capacity for concentrative amino acid accumulation; Conflux 2 (released by OH− or spermine without acyl-oxygen bond cleavage) originates from ion-exchanging sites which are key intermediates in reversed transport connected to the loci of Conflux 1 and probably also to sites of both cell wall and protein synthesis; Conflux 3 (released by trichloroacetic acid after a prior extraction with the solvent mixture) containing alanine, lysine, and some peptides, is of unknown relationship to the other confluxes; Conflux 4 (released by NH2OH or OH− with acyl-oxygen bond fission) consisting primarily of aspartic acid, but also containing some leucine, indicates the existence of an unusual, labile, aspartic acyl complex in the cell periphery. However, with regard to Conflux 4, dilute NH4OH, which releases alanine as the amide from a labile linkage in the teichoic acids, did not release leucine amide or isoasparagine in these experiments, although it strongly promoted reserved transport. 3. 3. The confluxes are compared with the internal and expandable amino acid pools reported in other organisms, and the implications of the heterogeneous metabolic origins and destinations of the confluxes for interpreting the physiological functions of the pools are discussed.

Very slow growth of Paracoccus denitrificans in a recycling fermentor

conic acid. In this case, the only role o f the enzyme might lie in energy production. Indeed, addition of glucose to carbonand energy-limited chemostat cultures results in a significant increase in cell yields with the concomitant production of gluconic acid. Carbon-limited chemostat cultures of A. calcoaceticus, growing on acetate, ethanol or succinate, contain appreciable constitutive levels of glucose dehydrogenase. The enzyme is associated with the cytoplasmic membrane but it can be solubilized with detergent and purified with a simple ionexchange step (Duine et al., 1979, 1982). The coenzyme, pyrrolo-quinoline-quinone (PQQ), is dissociable from the enzyme so that an apoenzyme can be prepared from it (Duine et al., 1979). Bivalent ions like Ca 2+ and Mg 2+ highly stimulate the recombination process of apoenz~rne and PQQ to an active holoenzyme. Screening a mutagenized culture of A. calcoaceticus on agar plates containing glucose and a pHindicator, yielded several non-acid-producing mutan ts which are impaired in the biosynthesis of PQQ. Addition of PQQ to cultures of these mutan ts restored the capacity to oxidize glucose to gluconic acid.