Changes in the concentration of inorganic phosphate (Pi) sugar phosphate, phosphoenol pyruvate (PEP), the adenylate charge and pH were followed by 31P-NMR and assay of cell extract during glucose fermentation in Brochothrix thermosphacta. These changes provide an estimation of glycolytic flux via pyruvate kinase as opposed to cycling of PEP through the phosphotransferase system. There appears to be a strong causal relationship between the reduced pH, the high NTP level and the apparent reduction in pyruvate kinase activity, which support the contention that synergistic effect of pH and ATP on pyruvate kinase is an important regulatory factor in these cells.
The nitrogenase activities of enterobacteria isolated from sugarcane roots and sugarcane waste products were usually 100-400nmol C2H4/culture/h. The isolates were characterized both phenotypically and chromosomally. Four phenotypic groups were differentiated. Group 1 isolates were identified as Klebsiella pneumoniae by computer-aided Microbact System 24 E and showed over 70% DNA homology with Klebsiella pneumoniae UQM 90. Isolates of group 3 and group 4 were identified as Enterobactercloacae and Enterobacter agglomerans respectively by the Microbact 24 E, but DNA-DNA hybridization studies suggested that isolates of these 2 groups belonged to the same species and did not belong to either Enterobactercloacae or Enterobacter agglomerans. Group 2 isolates were identified as Enterobacter agglomerans by the Microbact 24 E, but G+C% and DNA-DNA hybridization studies contradicted this identification. The G +C% values and phenotypic characteristics indicated that group 2 isolates might represent a new species of the genus Klebsiella while the isolates of group 3 and group 4 together could constitute a new species of the genus Enterobacter.
In C. murisepticum, a constitutive extracellular invertase hydrolyzes raffinose to melibiose and fructose. During growth on raffinose plus glucose, an unexpected appearance of sucrose was discovered in the growth medium. The enzyme preparation from raffinose-grown culture supernatant could synthesize in vitro, sucrose, and difructose from raffinose plus glucose or difructose from raffinose alone. The results thus bring to light a novel attribute of the extracellular invertase, of catalyzing the transfer of the fructose moiety of raffinose to glucose and fructose, thereby achieving the synthesis of sucrose, and difructose, respectively.
To elucidate the taxonomic position of "Rhodospirillum centenum" IAM 14193T (=ATCC 43720T) (T=type strain), a cyst-forming anoxygenic phototrophic bacterium, the small rRNA sequences (about 1, 300 bases) were determined for the organism, two cyst-forming isolates (MT-SP-2 and MT-SP-3), Rhodopseudomonas palustris ATCC 17001T and Erythrobacterlongus JCM 6170T by using the reverse transcription-dideoxy sequencing method. "Rhodospirillum centenum" IAM 14193T and the two isolates had high phylogenetic affinity (98.8% homology). The sequences of the two isolates were identical. Comparative analysis of the presently determined sequences and the reference sequences (1, 107 bases) revealed that the three cyst-forming organisms belonged to the α group of the class Proteobacteria, and were distantly related to Rhodospirillumrubrum (the type species, α-1 subgroup) (89.9% homology) and the representative members of other subgroups (α-2, -3 and -4) (87.6-89.8% homology). They constituted a new phylogenetic branch in the α group. "Rhodospirillum centenum" IAM 14193T possessed Q-9 as the major quinone but no rhodoquinones or menaquinones, that was a quinone system distinct from that of other anoxygenic phototrophs. The phylogenetic evidence and the phenotypic characteristics indicated that "Rhodospirillumcentenum" IAM 14193T did not belong to the genus Rhodospirillum. We proposed Rhodocista centenaria gen. nov., sp. nov. for the organism.
Mass spectrometry confirmed that the entire denitrification system of Thiosphaera pantotropha is present under aerobic conditions. Acetate-dependent oxygen uptake is totally inhibited by 0.3mM cyanide. Nitrate reduction by cells grown with and without nitrate was inhibited by 10 and 15mM azide, respectively. The nitrous oxide reductase was totally inhibited by 1% acetylene or 0.125mM cyanide. This reductase was only temporarily inhibited by 10mM azide. Frozen/thawed cell suspensions of T. pantotropha produced, under anaerobic conditions, only nitric oxide. The effects of various inhibitors on T. pantotropha are summarized and considered in relation to their effect on other denitrifiers.
Eight strains of Leucosporidium fellii, L. lari-marini, Filobasidium capsuligenum, F. floriforme, and F. uniguttulatum were examined for partial base sequences in positions 492-625 (134 bases) of 26S rRNA and in positions 1451-1618 (168 bases) of 18S rRNA. In the 26S rRNA partial base sequence, there were low maximum homologies (46-58%) among L. fellii, L. lari-marini, and Filobasidium species. In the 18S rRNA partial base sequence, F. capsuligenum had 4 and 5 base differences, compared with F. floriforme and F. uniguttulatum, respectively. Leucosporidiumlari-marini and L. fellii showed 0 and 4 base differences with Cystofilobasidiumcapitatum and L. scottii, respectively. The sequence data obtained were discussed phylogenetically and taxonomically, especially on transferring L. lari-marini to the genus Cystofilobasidium.
The growth rates of Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes, which are known to be sensitive to low pH, were decreased to less than half of the maximum rates at extracellular pH (pHe) 6.0, and their growth rates were extremely low at pHe 5.6. Their intracellular pH(pHi) was decreased linearly as pHe was lowered from 6.8 to 6.0, irrespective of the presence or absence of cellobiose. However, pHi was not decreased markedly as pHe was lowered from 6.0 to 5.6 when cellobiose was given, resulting in the increased pH gradient across the cell membrane (ΔpH). On the other hand, when cellobiose was not supplied, pHi was still linearly decreased with the decrease of pHe to 5.6. These results suggest that much energy is needed to form ΔpH at pHe 5.6. Membrane potential (Δψ) and proton motive force (PMF) were not significantly affected by the change in pHe. Addition of 3% ethanol markedly decreased the ΔpH of these bacteria at pHe 5.6. In R. flavefaciens and F. succinogenes, Δψ was notably decreased by 3% ethanol, while in R. albus Δψ was slightly decreased by ethanol. Compared with Megasphiaera elsdenii, which is relatively tolerant to low pH, these cellulolytic bacteria appear to have low capacity to maintain their pHi against the decrease in pHe.
Polyamines of thermophilic Gram-negative eubacteria, "Rhodothermusmarinus" ATCC 43812, Thermus sp. ATCC 43814 and Thermonemalapsum ATCC 43542 were analyzed by high-perfomance liquid chromatography and gas chromatography-mass spectrometry. "R. marinus" contained spermidine, spermine, thermopentamine, a tertiary tetraamine (N4-aminopropylspermidine) and a quaternary pentaamine (N4-bis(aminopropyl) spermidine). Thermus sp. ATCC 43814 contained putrescine, cadaverine, norspermidine, spermidine, homospermidine, norspermine, spermine, thermospermine, aminopropylhomospermidine, caldopentamine, agmatine, two tertiary tetraamines (N4-aminopropylnorspermidine and N4-aminopropylspermidine) and two quaternary pentaamines (N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine). Homospermidine and homospermine were detected in Thermonemalapsum as the major polyamine. These distribution patterns of long and branched polyamines are distinctive in the thermophiles, indicating that unusual polyamine profiles serve to estimate chemotaxonomic and phylogenetic relationships within thermophilic eubacteria.
Eleven strains of species in the apiculate yeast genera Hanseniaspora, Nadsonia, and Saccharomycodes were examined for partial base sequence determinations of 18S and 26S rRNAs. In the partial base sequence of 26S rRNA (positions 493-622, 130 bases), percent similarities were 65-79, 76-84, and 70-76 between the genera Hanseniaspora and Nadsonia, the genera Hanseniaspora and Saccharomycodes, and the genera Nadsonia and Saccharomycodes, respectively. These apiculate yeasts showed 72-85 percent similarity with S. cerevisiae. In the partial base sequences of 26S rRNA (positions 1611-1835, 225 bases) and of 18S rRNA (positions 1451-1618, 168 bases), the number of base differences was calculated to be 38-26, 27-9, and 30-23, and 12-8, 11-5, and 13-8 between the above-mentioned genera, respectively. These apiculate yeasts showed 33-9, and 12-4 base differences, respectively, with S. cerevisiae. The three apiculate yeast genera were recognized phylogenetically based on the sequence data obtained. Some discussions were made, especially on dividing the members of the genus Hanseniaspora into two groups at the generic level.
Restriction fragment length polymorphism (RFLP) of DNA was examined for six species of mushrooms in the genus Pleurotus: P. citrinopileatus, P. cornucopiae, P. ostreatus, P. pulmonarius, P. salmoneostramineus and P. sajor-caju. DNAs prepared from the species were analyzed on agarose gels after digestion with restriction enzymes such as HaeIII, HinfI, MspI, RsaI, Sau3AI and StyI. RFLP banding patterns generated by each of the six enzymes were found to be species-specific. The banding patterns of P. citrinopileatus and P. cornucopiae were distinctive but relatively similar among six species examined, indicating that the two species were closely related. Six isolates of P. cornucopiae, three wild and three commercial strains, were also analyzed. Based on the RFLP patterns, the wild isolates were found to be closely related to P. citrinopileatus, while the commercial strains were to P. cornucopiae.
A process of continuous citric acid production from sugarcane molasses by fermentation with immobilized whole cells of A. niger KCU 520 is described. Both calcium alginate beads and polyacrylamide gel (PAG) slab entrapment methods were used for immobilization of cells. The optimum fermentation conditions for citric acid fermentation by immobilized cells were sugars, 10%; pH, 4.0; inoculum size, 20%, and temperature, 32-35°C. With calcium-alginate and PAG-immobilized cells, an overall citric acid production rate of 8.5g and 12.0g/l/day (9.3% and 12% sugar conversion to citric acid per day), respectively was achieved in a single-stage bioreactor. When the PAG-immobilized cells were used in a two-stage bioreactor, approximately 20.0g citric acid/l/day (20% sugar conversion to citric acid per day) was produced and the PAG-immobilized cells were continuously used for at least 20 days without any significant loss of productivity.
In laboratory study, some factors influencing the secretion of alginic acid by highly mucoid strain of Azotobacter vinelandii were investigated in batch culture. The highest alginate yields were (5.5-6.2mg ml-1 culture supernatant) obtained for growth in a nitrogen-rich and phosphate-limited medium with sucrose as carbon source, aerated by shaking at 240 rpm. At 140rpm, alginate yield was only 1.6mg ml-1. If sucrose was replaced by glucose, both growth and alginate production ware reduced.