Coflocculation of Escherichia coli and Schizosaccharomyces pombe.

Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S. pombe showed much less coflocculation than the other yeasts (Peng et al. 2001)). S. pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1delta (Man:Gal=1:0). The wild-type induced very low levels of coflocculation (3%) while gms1delta induced a remarkable amount of coflocculation (48%). Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose. The S. cerevisiae mnn2 mutant, with a mannan structure similar to gms1delta, also showed a high degree of coflocculation (40%). However, S. cerevisiae mutant mnn9, with a mature core similar to S. pombe, showed decreased coflocculation (21.3%). Both these S. cerevisae mutants were sensitive to mannose inhibition. Coflocculation of E. coli and gms1delta also could be inhibited by gms1delta mannan and plant lectins, such as HHA, GNA and NPA, specific to either alpha-1-3- or alpha-1-6-linked mannosyl units. From these results we conclude that the E. coli lectins may have specificity for alpha-1-6- and alpha-1-3-linked mannose residues either in the outer chain or in the core of S. pombe, but in wild-type strains these mannose residues are shielded by galactose residues.

Heterologous expression of the Bacillus pumilus endo-beta-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae.

The endo-beta-xylanase-encoding gene (xynA) of Bacillus pumilus PLS was isolated from a genomic DNA library and the open reading frame (ORF) was inserted in expression vectors for the yeast Saccharomyces cerevisiae. Plasmid pFN3 harboured the xynA ORF fused to the yeast mating pheromone alpha-factor signal sequence (MFalpha1s) under the control of the alcohol dehydrogenase II gene promotor (ADH2P) and terminator (ADH2T) sequences. In plasmid pFN4, the MFalpha1S-xynA ORF was brought under the control of the phosphoglycerate kinase I gene promotor (PGK1p) and terminator (PGK1T) sequences. Autoselective, recombinant S. cerevisiae [fur1::LEU2] strains bearing pFN3 or pFN4 secreted functional endo-beta-xylanase when grown in complex medium. Enzymatic activities in the culture supernatants reached maximum levels of 8.5 nkat/ml and 4.5 nkat/ml, respectively. The temperature and pH optimum for both the bacterial and the recombinant xylanase were 58 degrees C and pH 6.2.

Flocculation and coflocculation of bacteria by yeasts.

Biotransformations in natural environments frequently involve interactions between microorganisms. Although there are many reports on the interactions between bacteria, interactions between yeasts and bacteria have not been extensively studied. Previously we reported on the flocculation and coflocculation of Pediococcus damnosus by Saccharomyces cerevisiae. Now we report that several other yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, and Schizosaccharomyces pombe, induce flocculation with several industrially or medically relevant bacteria, including Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Candida utilis was one of the best flocculation inducers. The results are discussed with respect to interactions between yeasts and bacteria and their applications in industry and medicine.

Decrease in cell surface galactose residues of Schizosaccharomyces pombe enhances its coflocculation with Pediococcus damnosus.

Pediococcus damnosus can coflocculate with Saccharomyces cerevisiae and cause beer acidification that may or may not be desired. Similar coflocculations occur with other yeasts except for Schizosaccharomyces pombe which has galactose-rich cell walls. We compared coflocculation rates of S. pombe wild-type species TP4-1D, having a mannose-to-galactose ratio (Man:Gal) of 5 to 6 in the cell wall, with its glycosylation mutants gms1-1 (Man:Gal = 5:1) and gms1Delta (Man:Gal = 1:0). These mutants coflocculated at a much higher level (30 to 45%) than that of the wild type (5%). Coflocculation of the mutants was inhibited by exogenous mannose but not by galactose. The S. cerevisiae mnn2 mutant, with a mannan content similar to that of gms1Delta, also showed high coflocculation (35%) and was sensitive to mannose inhibition. Coflocculation of P. damnosus and gms1Delta (or mnn2) also could be inhibited by gms1Delta mannan (with unbranched alpha-1,6-linked mannose residues), concanavalin A (mannose and glucose specific), or NPA lectin (specific for alpha-1,6-linked mannosyl units). Protease treatment of the bacterial cells completely abolished coflocculation. From these results we conclude that mannose residues on the cell surface of S. pombe serve as receptors for a P. damnosus lectin but that these receptors are shielded by galactose residues in wild-type strains. Such interactions are important in the production of Belgian acid types of beers in which mixed cultures are used to improve flavor.

Sensitivity of Saccharomyces cerevisiae to tannic acid is due to iron deprivation.

Tannic acid inhibited the growth of the yeast Saccharomyces cerevisiae. Growth medium supplementation with more nitrogen or metal ions showed that only iron ions could restore the maximal growth rate of S. cerevisiae. Tannic acid resistant mutants were previously isolated by screening for tannic acid resistance and were all cytoplasmic petite mutants. While the wild type was very sensitive to iron deprivation conditions when grown in aerobic conditions, the mutants, whether grown aerobically or anaerobically, showed the same growth rate under iron-limited conditions as under iron-repleted conditions. Also, the wild type grown anaerobically was not affected by iron-limited conditions. Cytoplasmic petite mutants obtained by ethidium bromide mutagenesis behaved like the other mutants. During iron limitation, the wild type showed a reduced oxygen uptake rate. Maximal growth rate of the wild type in iron-limited conditions could be restored by the addition to the media of unsaturated fatty acids and sterol. Iron deprivation caused by tannic acid may thus affect the synthesis of a functional respiratory chain as well as the synthesis of unsaturated fatty acids and (or) sterol.

Cloning and sequencing of the LEU2 homologue gene of Schwanniomyces occidentalis.

A gene that complements the leu2 mutation of Saccharomyces cerevisiae has been cloned from Schwanniomyces occidentalis. The gene codes for a protein of 379 amino acids. As expected for a Schwanniomyces gene, it has a high AT content, which is also reflected in the codon usage. The sequence homology with other known leu2 complementing genes is low.

Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene.

In the preceding paper (Derom et al., 1981) we described the cloning in bacterial plasmids of the simian virus 40 (SV40) small-t antigen gene under transcriptional control of the bacteriophage lambda pL promoter. Systematic variation of the distance and/or nucleotide sequence between the Shine-Dalgarno ribosome interaction sequence and the small-t translation initiation codon leads to considerable differences in production of small-t by the different plasmids. Secondary structure models derived for the different mRNAs confirm our previous conclusions about the requirement first for an accessible start codon and second for an accessible ribosome interaction site for efficient translation initiation. Secondary structure models for mRNAs from plasmids containing the small-t gene under control of the lac promoter are in agreement with these conclusions.

Secondary structure of mRNA and efficiency of translation initiation.

A series of recombinant plasmids, containing the cro gene of phage lambda, exhibit strikingly different levels of expression depending apparently only on the nucleotide sequence of the untranslated 5' mRNA (Roberts et al., 1979). We postulate that initiation of translation involves interaction between an activated 30S ribosomal subunit and the 5'-terminal region of a messenger RNA already folded in a specific secondary structure. The observed variation in cro synthesis can then adequately be explained by secondary structure models which were derived for the different mRNAs. To maximize expression, it appears necessary that the initiation codon and, although less important, the ribosome interaction site are accessible.

Characterization of the major altered leader sequence of late mRNA induced by SV40 deletion mutant d1-1811.

d1-1811 is a viable SV40 mutant with a 40 base pair deletion that includes the major wild-type capping site of late mRNA at map position 0.72. The late viral mRNAs induced by d1-1811 have now been further characterized by inversed S1-mapping analysis. The S1-resistant, 32P-labeled RNA fragments derived from the leader region were examined by fingerprinting and by analysis of RNase T2-generated 5'-terminal cap structures. The results show that most if not all of the mutant leader fragments analyzed have their 5' terminus to the left of the d1-1811 deletion site, i.e., closer to the origin of DNA replication. The major altered leader fragment is a continuous transcript from the DNA in the region 0.716 to 0.761 map unit and its 5' terminus has been precisely mapped at nucleotide L290. The observation that the cap structure of the major altered leader is only a minor cap species in wild-type late RNA suggests regulation in the use of different capping sites in SV40.

Secondary structure of the 5' end of bacteriophage MS2 RNA Methoxyamine and kethoxal modification.

To refine the secondary structure model of the 5' end of the bacteriophage MS2 genome, 32P-labeled MS2 RNA was partially digested with T1 RNase or with Cm-RNase and the 5'-end fragment was isolated, renatured and submitted to treatment with methoxyamine or kethoxal. The resulting modified RNA was digested with T1 RNase and the products were separated by minifingerprinting. Methoxyamine-induced modification of exposed cytidines was detected by differential mobility of modified oligonucleotides, while kethoxal-induced alteration of exposed guanosines was monitored by resistance to T1 ribonuclease digestion. The positions of the modified residues are discussed in terms of an improved secondary structure model proposed for the 5' end of the viral RNA. The structure itself is discussed in relation to sequence conservation and biological function.

Asymmetric linker molecules for recombinant DNA constructions.

Asymmetric EcoRI DNA linkers consisting of an AATTC(A)7 dodecamer and a complementary G(T)7 octamer were synthesized. Ligation of such linkers to DNA fragments obviates the need for EcoRI digestion prior to cloning in EcoRI-cleaved vectors.

Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene.

Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.