An in vitro study was conducted to examine the metabolism of phenylalanine (Phe) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39°C for 12h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. The net degradation rate (μmol/g microbial nitrogen) of Phe in B was about 1.5-fold higher than that in P. Phe was converted mainly into phenylacetic acid (PAA) and unknown compound(s) that presumably involved tyrosine in B, P, and BP during the 12h incubation period. Small amounts of benzoic acid (BZA), and traces of phenylpropionic acid (PPR) and phenyllactic acid (PLA) were also produced from Phe. PAA production in B was found to be higher than that in P, whereas it was significantly higher in BP. Although BZA production was less than one-tenth that of PAA production, it was higher in P than in B and BP. PPR was detected in both B and BP, but not in P. PLA was detected only in B. The production of unknown compound(s) was higher in B than in P and BP.
Phenylalanine (Phe) synthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and a combination of the two mixture (BP) in an in vitro system were quantitatively investigated using phenylpyruvic acid (PPY) and phenylacetic acid (PAA) as substrates. Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicagosativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39°C for 12h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. A large quantity of Phe was produced from both PPY and PAA not only in B but also in P. In B suspensions, free Phe also accumulated in the medium only when PPY was used as a substrate. The ability of B to synthesize Phe from both PPY and PAA (expressed as unit ‘per microbial nitrogen’) was 5.1 and 24.8% higher than P. respectively. Phe production from PPY in B and P was 43.5 and 55.2% higher than that from PAA. Large amounts of PAA (17-27%) were produced from PPY in all microbial suspension and production amounts were similar in B and P. Small amounts of benzoic acid (BZA) were produced from PPY and PAA in B, P, and BP, and higher BZA production was observed in P as compared to B. Phenylpropionic acid (PPR) was produced in B from both PPY and PAA, but not in P or BP. A trace amount of phenyllactic acid (PLA) was detected only from PPY in B. Higher concentrations of an unknown compound from PPY and PAA were found to be accumulated in the body protein of B and also in the medium of P, and production of the compound from both PPY and PAA was also higher in B than P.
RNA polymerase was purified from the unicellular cyanobacterium, Synechococcus sp. strain PCC 7942, and found to be associated with a 52 kilodalton (kDa) polypeptide. The determined N-terminal sequence of the polypeptide was identical to the predicted amino-acid sequence of the rpoD1 gene product. Furthermore, the rpoD1 gene is suggested to be indispensable for viability by the inability to disrupt the gene. These results indicate that the rpoD1 gene product is the principal sigma factor of RNA polymerase.
Thirteen Armillaria isolates, collected from various geographical areas in tropical Africa and previously characterized by cultural morphology, pairing tests and isozyme analysis, were evaluated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). DNA regions corresponding to the intergenic spacer (IGS) and internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The IGS amplification products were about 875bp long and uniform in length among the isolates. The amplified-ITS region showed two different lengths corresponding to two groups. The first group included the isolates believed to belong to A. mellea ssp. africana and two Kenyan isolates (K11 and K12) belonging to a yet unnamed biological species. The second group included isolates identified as A. heimii and a Tanzanian isolate (T7). Each length variant of the ITS showed distinct RFLP banding patterns. Digestion with EcoRI confirmed the two polymorlphic groups while the endonucleases Alul and Ndell discriminated the A. mellea isolates from the Kenyan isolates K11 and K12. In addition, the latter enzyme showed a slight dissimilarity between the A. heimii isolates from Western and Eastern Africa (C1 and Z1). Digestion with Hinfl cleaved the isolates of A. heimii into two sub-groups corresponding to the heterothallic and homothallic forms. This endonuclease also indicated that the isolate T7, originating from Tanzania, was clearly similar to the heterothallic species A. heimii. Data presented support the maintenance of three distinct species of Armillaria in tropical Africa with A. heimii as a variable species, the isolates of which were separated in accordance with their sexual system. The results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for the ecological studies of Armillaria species.
Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including β-1, 3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and β-1, 3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, , T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-β-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.
In Japan, high-sugar fermented vegetable extracts are novel functional food products for which sugar-tolerant yeasts are employed during processing. In order to understand the yeast distribution in these foods and their role in the functionality of such foods, we isolated sugar-tolerant yeasts from nine sample products, together with one sample each of fermented extract of ume (Japanese apricot) and honey. Twenty-three strains were identified as Zygosaccharomyces rouxii; one strain as Z. bailli; one strain as Torulaspora delbrueckii; and one strain as Candida bombicola. Nearly 90% of the identified strains belonged to Z. rouxii with variations in fermentation and assimilation properties. All strains grew well on 50% w/w glucose medium, and all but two strains grew on 60% w/w glucose medium. Sixteen strains belonged to the strong sugar tolerance type (poor or no growth at 1% and maximum growth at 30 or 40% w/w glucose); four strains to the moderate type (grew well at 1% and maximum growth at 10 or 20% w/w glucose); and seven strains to the weak type (maximum growth only at 1% w/w glucose). One strain of Z. rouxii, V19, grew up to 80% (w/w) glucose in liquid medium. In view of salt tolerance, only two strains belonged to the moderate type (maximum growth at 0.5 or 1M NaCl); the remaining strains all belonged to the weak type (maximum growth only at 0M NaCl). This suggests that sugar tolerance and salt tolerance of yeasts have different aspects.
Polyamines of the four families and the five related genera within the gamma subclass of the class Proteobacteria were analyzed by HPLC with the objective of developing a chemotaxonomic system. The production of putrescine, diaminopropane, cadaverine, and agmatine are not exactly correlated to the phylogenetic genospecies within 36 strains of the genus Aeromonas (the family Aeromonadaceae) lacking in triamines. The occurrence of norspermidine was limited but not ubiquitous within the family Vibrionaceae, including 20 strains of Vibrio, Listonella, Photobacterium, and Salinivibrio. Spermidine was not substituted for the absence of norspermidine in the family. Agmatine was detected only in Photobacterium. Salinivibrio and some strains of Vibrio were devoid of polyamines. Vibrio ("Moritella") marinus contained cadaverine. Within the family Pasteurellaceae, Haemophilus contained cadaverine only and Actinobacillus contained no polyamine. Halomonas, Chromohalobacter, and Zymobacter, belonging to the family Halomonadaceae, ubiquitously contained spermidine and sporadically cadaverine and agmatine. Shewanella contained putrescine and cadaverine; Alteromonas macleodii, putrescine, 2-hydroxyputrescine, cadaverine, 2-hydroxyspermidine, and spermidine; Pseudoalteromonas, putrescine, cadaverine, and spermidine; Marinobacter, spermidine; and Marinomonas, putrescine and spermidine. Their polyamine profiles serve as a chemotaxonomic marker within the gamma subclass.
Since Bifidobacterium bifidum, one of the strains of medical preparations used for human intestinal disorders, is sensitive to rifampicin (RFP) and fluoroquinolones, its therapeutic effect cannot be guaranteed when it is administered concomitantly with these antibiotics. To develop new strains of B. bifidum that are resistant to these drugs, B. bifidum RFR61, which is highly resistant to RFP, was selected by the N-methyl-N′-nitrosoguanidine (MNNG) mutation method. Then, B. bifidum OFR9 was selected in vitro from B. bifidum RFR61 by serial passage to increasing concentrations of ofloxacin (OFLX) on a solid medium. The minimal inhibition concentrations (MIC) of RFP and fluoroquinolones for B. bifidum OFR9 were>256μg/ml and 16-256μg/ml, respectively. We investigated the effects of B. bifidum OFR9 on the fecal bacterial flora of mice administered with both antibiotics and B. bifidum OFR9. The results showed that the concurrent use of B. bifidum OFR9 and antibiotics prevented the decrease of bifidobacteria, and quickly restored the flora to normal as compared with the use of antibiotic or parent strain therapy alone. The survival of Shigella organisms in mouse feces rapidly decreased, and were removed within two days as a result of the oral administration of B. bifidum OFR9.