Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH.

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH4OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.

A multi-gene phylogeny provides additional insight into the relationships between several Ascosphaera species.

Ascosphaera fungi are highly associated with social and solitary bees, with some species being pathogenic to bees (causing chalkbrood) while others are not, and proper identification within this genus is important. Unfortunately, morphological characterizations can be difficult, and molecular characterizations have only used one genetic region. We evaluated multiple phylogenies of the Ascosphaera using up to six loci: the Internal Transcribed Spacer (ITS) region, 18S rRNA, 28S rRNA, Elongation Factor-1α (EF-1α) the RNA polymerase II largest subunit (RPB1), and the second largest subunit (RPB2). The ITS sequence alone produced an inadequate phylogeny, and the addition of both the 18S and 28S rRNA loci to the ITS sequence produced a phylogeny similar to that based on all six genetic regions. For all phylogenies, Ascosphaera torchioi was in a separate clade that was the most basal, with a strong genetic similarity to Eremascus albus, introducing the possibility of paraphyly within Ascosphaera. Also, based on this new phylogeny, we now suggest that the Apis mellifera (honey bee) pathogens arose within a group of saprophytes, and the Megachile (leafcutting bees) pathogens arose separately.

Protein-protein interactions among the components of the biosynthetic machinery responsible for exopolysaccharide production in Streptococcus thermophilus MR-1C.

AIM: This study identified protein-protein interactions among the biosynthetic machinery responsible for exopolysaccharide (EPS) production in Streptococcus thermophilus MR-1C. METHODS AND RESULTS: Protein-protein interactions were investigated using the yeast two-hybrid system. A strong protein-protein interaction was detected between the transmembrane activation protein Wzd and the protein tyrosine kinase Wze. Weaker protein-protein interactions were detected between two duplicate Wze proteins and between Wze and the phosphotyrosine phosphatase Wzh. Protein-protein interactions involving a Wzd/Wze fusion protein and Wzd and Wze may indicate that these proteins form multi-protein complexes. All combinations of the Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation) and CpsU (membrane translocation) were analysed for protein-protein interactions but no additional interactions were discovered using the yeast two-hybrid system. CONCLUSIONS: Interactions among the phosphotyrosine phosphatase, tyrosine kinase, and transmembrane activation protein are important in the regulation of capsule biosynthesis in Strep. thermophilus MR-1C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some valuable insight into the organization and interactions between the many proteins involved in EPS production. A better understanding of this process may facilitate the genetic manipulation of capsule production to impart desirable properties to dairy starter cultures.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains.

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

A polymorphic, prespore-specific cell surface glycoprotein is present in the extracellular matrix of Dictyostelium discoideum.

Polymorphisms of a major developmentally regulated prespore-specific protein (PsA) in Dictyostelium discoideum slugs are described. These polymorphisms allowed discrimination between PsA (found on the cell surface and in the extracellular matrix) and a similar extracellular but nonpolymorphic protein, ShA. The two proteins were also distinguished by their differing reactivities with a range of monoclonal antibodies and by their sensitivity to release from the sheath with cellulase. The results are discussed in terms of the molecular and genetic relationships between the cell surface and the extracellular matrix during development.

Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1.

All of the plasmid-carried genes expressed during vegetative growth are essential for long-term maintenance of plasmid Ddp1 in the nucleus of Dictyostelium discoideum. Deletion of Ddp1 genes expressed only during development had no detectable effect on plasmid maintenance. Deletion of vegetatively expressed genes, either singly or in pairs, resulted in (i) a rapid loss of plasmid from cells grown in the absence of selection for plasmid retention, (ii) variation in the proportion of monomer to multimer forms of the plasmid molecules, and/or (iii) abnormalities in plasmid copy number. At least two plasmid-encoded gene products influence patterns of expression of plasmid genes.

Inheritance of extrachromosomal ribosomal DNA during the asexual life cycle of Dictyostelium discoideum: examination by use of DNA polymorphisms.

Wild-type isolates of Dictyostelium discoideum exhibited differences in the size of restriction fragments of the extrachromosomal 88-kilobase ribosomal DNA (rDNA) palindrome. Polymorphisms in rDNA also were found among strains derived solely from the NC4 wild-type isolate. These variations involved EcoRI fragments II, III, and V; they included loss of the EcoRI site separating fragments II and V and deletion and insertion of DNA. More than one rDNA form can coexist in the same diploid or haploid cell. However, one or another parental rDNA tended to predominate in diploids constructed, using the parasexual cycle, between haploid NC4-derived strains and haploid wild-type isolates. In some cases, most if not all of the rDNA of such diploids were of one form after ca. 50 generations of growth. Segregant haploids, derived from diploids that possessed predominantly a single rDNA allele, possessed the same allele as the diploid and did not recover the other form. This evidence implies that replication does not proceed from a single chromosomal or extrachromosomal copy of the rDNA during the asexual life cycle of D. discoideum.

Presence of nuclear associated plasmids in the lower eukaryote Dictyostelium discoideum.

High copy number plasmids have been identified in six out of 25 wild-type strains of the cellular slime mould Dictyostelium discoideum, a model organism in developmental biology (Loomis, 1982). The characterization of three plasmids, from the NC4 (Ddp1), WS380B (Ddp2) and OHIO (Ddp3) wild isolates, is presented here. We show that they are nuclear associated and non-homologous to the mitochondrial DNA and extrachromosomal ribosomal DNA.

The discoidin I gene family of Dictyostelium discoideum is linked to genes regulating its expression.

The discoidin I protein has been studied extensively as a marker of early development in the cellular slime mold Dictyostelium discoideum. However, like most other developmentally regulated proteins in this system, no reliable information was available on the linkage of the discoidin genes to other known genes. Analysis of the linkage of the discoidin I genes by use of restriction fragment length polymorphisms revealed that all three discoidin I genes as well as a pseudogene are located on linkage group II. This evidence is consistent with the discoidin I genes forming a gene cluster that may be under the control of a single regulatory element. The discoidin I genes are linked to three genetic loci (disA, motA, daxA) that affect the expression of the discoidin I protein. Linkage of the gene family members to regulatory loci may be important in the coordinate maintenance of the gene family and regulatory loci. A duplication affecting the entire discoidin gene family is also linked to group II; this appears to be a small tandem duplication. This duplication was mapped using a DNA polymorphism generated by insertion of the Tdd-3 mobile genetic element into a Tdd-2 element flanking the gamma gene. A probe for Tdd-2 identified a restriction fragment length polymorphism in strain AX3K that was consistent with generation by a previously proposed Tdd-3 insertion event. A putative duplication or rearrangement of a second Tdd-2 element on linkage group IV of strain AX3K was also identified. This is the first linkage information available for mobile genetic elements in D. discoideum.

Linkage Analysis of Nystatin Resistance Mutations in DICTYOSTELIUM DISCOIDEUM.

Earlier linkage analyses of nystatin resistance loci in Dictyostelium discoideum tentatively mapped the nysB and nysC loci to the previously unmarked linkage group V. The data presented here establishes that nysB maps to linkage group VI and that nysC maps to linkage group IV. The third nystatin resistance locus, nysA, maps to linkage group II.

Translocations in Dictyostelium discoideum.

Fourteen translocations of independent origin were identified in Dictyostelium discoideum on the basis of segregation anomalies of diploids heterozygous for these chromosome rearrangements, all of which led to the cosegregation of unlinked markers. Many of these translocations were discovered in strains mutagenized with MNNG or in strains carrying mutations affecting DNA repair; however, spontaneous translocations were also obtained. Haploid mitotic recombinants of the rearranged linkage groups were produced from diploids heterozygous for the translocations at frequencies of up to 5% of viable haploid segregants; this is at least a ten-fold higher frequency than that seen with diploids not heterozygous for translocations (approximately 0.1%). These haploid recombinants included both translocated and nontranslocated strains. The T354(II, VII) translocation and possibly the T357(IV, VII) translocation reduce the chromosome number to n = 6; haploids carrying 11 other translocations all have karyotypes with n = 7. Genetic characterization of the T357(IV, VII) translocation showed that the bwnA and whiC loci normally found on linkage group IV were physically linked to the linkage group VII loci couA, phgA, bsgB and cobA.

Recessive lethal mutations and the maintenance of duplication-bearing strains of Dictyostelium discoideum.

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.

A genetic map of Dictyostelium discoideum based on mitotic recombination.

A genetic map of the cellular slime mold Dictyostelium discoideum is presented in which 42 loci are ordered on five of the seven linkage groups. Although most of the loci were ordered using standing mitotic crossing-over techniques in which recessive selective markers were employed, use was also made of unselected recombined haploid strains. Consistent with cytological studies in which the chromosomes appear to be acrocentric, only a single arm has been found for each of the five linkage groups studied. The mating-type locus, matA, has been located in the tsgE-sprA interval on linkage group I on the basis of studies on diploids formed between strains of opposite mating type that have escaped from vegetative incompatibility.

The use of DNA probes for taxonomic study of dictyostelium wild isolates.

The classification of 27 wild isolates assigned to Dictyostelium discoideum on the basis of morphological criteria was reexamined using probes specific for DNA sequences cloned from the type strain NC4. These probes included ones specific for ribosomal spacer DNA regions and for a ribosomal RNA coding sequence, as well as probes for two chromosomal gene families (actin and discoidin) and for the DIRS-1 transposable element. Four isolates (AC4, WS526, WS584 and ZA3A) which had previously been shown to have unusual mating characteristics were distinctly different from other isolates. We interpret these differences as indicating that the four atypical isolates represent species other than D. discoideum. Probes for the ribosomal spacer DNA either did not hybridize to the DNA of these four isolates or had decreased levels of hybridization to EcoRI restriction fragments of different lengths to that observed with the type strain. With the discoidin probe, all isolates had DNA fragments that hybridized but AC4, WS526, WS584 and ZA3A lacked a pair of fragments that were conserved in NC4 and other isolates. With the actin probe, AC4, WS526, WS584 and ZA3A lacked numerous fragments that the other isolates shared with NC4. The DIRS-1 probes showed strong hybridization with ZA3A and weak hybridization to the other three isolates; however, the major EcoRI fragment in WS526 and WS584 was smaller than that in NC4 while ZA3A and AC4 had fragments of similar size to that in NC4.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic analysis of two mutations affecting thymidine metabolism in Dictyostelium discoideum.

The tmpA600 mutation confers thymidylate synthase deficiency and thymidine auxotrophy to Dictyostelium discoideum. The tdrA600 mutation enhances transport of thymidine and thereby reduces the auxotrophic requirement of tmpA600 strains. The tmpA locus maps to linkage group III. The tdrA600 mutation is dominant and cosegregates with both linkage groups IV and VI, possibly because of a translocation between the two. The tdrA600 allele is sufficient to allow efficient incorporation of exogenous [3H]thymidine or [3H]uridine into TCA-precipitable material and to sensitize the cell to the nucleoside-analog inhibitor, 5-fluorodeoxyuridine. These properties make the tdrA mutation useful for studies requiring labelling of DNA or RNA in vivo.

Integrated maps of the chromosomes in Dictyostelium discoideum.

Detailed maps of the six chromosomes that carry the genes of Dictyostelium discoideum were constructed by correlating physically mapped regions with parasexually determined linkage groups. Chromosomally assigned regions were ordered and positioned by the pattern of altered fragment sizes seen in a set of restriction enzyme mediated integration-restriction fragment length polymorphism (REMI-RFLP) strains each harboring an inserted plasmid that carries sites recognized by NotI, SstI, SmaI, BglI and ApaI. These restriction enzymes were used to digest high molecular weight DNA prepared from more than 100 REMI-RFLP strains and the resulting fragments were separated and sized by pulsed-field gels. More than 150 gene probes were hybridized to blots of these gels and used to map the insertion sites relative to flanking restriction sites. In this way, we have been able to restriction map the 35 mb genome as well as determine the map position of more than 150 genes to with approximately 40 kb resolution. These maps provide a framework for subsequent refinement.

Chromosome rearrangements in Dictyostelium discoideum.

A tandem duplication (D350(III,III] of the whiB to radB interval of linkage group III has been characterized. The gene order on the duplication-bearing chromosome is: centromere, whiB500, radB+, whiB+, radB24, bsgA5, acrC4. Slow-growing, duplication-bearing strains (yellow-spored, radiation-resistant) produced four classes of faster growing sectors involving the whiB and radB loci: white-spored, radiation-sensitive (whiB500, radB24); white-spored, radiation-resistant (whiB500, radB+); yellow-spored, radiation-sensitive (whiB+, radB24); and yellow-spored, radiation-resistant. The first three classes can be explained as the products of single recombination events in which one copy of the whiB to radB interval was lost. The yellow-spored, radiation-resistant sectors probably arose by mutation elsewhere in the genome, but alternatively may represent multiple recombination events or deletion of part of one copy of the duplicated region. Loss of the duplicated segment was enhanced by irradiation with ultraviolet light (254 nm). Heterozygosity for a DNA repair mutation at the radB locus may have been involved in the formation of the duplication. It is proposed that translocations are a major cause of nonrandom segregation patterns such as the cosegregation of unlinked markers in Dictyostelium discoideum. Translocations involving all known linkage groups are tabulated and DNA damage by N-methyl-N'-nitro-N-nitrosoguanidine is implicated in the formation of translocations in D. discoideum.

Chromosome Fragments in DICTYOSTELIUM DISCOIDEUM Obtained from Parasexual Crosses between Strains of Different Genetic Background.

The first aneuploid strains of Dictyostelium discoideum have been unambiguously characterized, using cytological and genetic analysis. Three independently isolated, but genetically similar, fragment chromosomes have been observed in segregants from diploids formed between haploid strains derived from the NC4 and V12 isolates of D. discoideum. Once generated, the fragment chromosomes, all of which have V12-derived centromeres, can be maintained in a NC4 genetic background. Genetic evidence is consistent with the view that all three fragment chromosomes studied encompass the region from the centromere to the whiA locus of linkage group II and terminate in the interval between whiA and acrA. From cytological studies, one of the fragment chromosomes consists of approximately half of linkage group II.-We observed no deleterious effect on viability or asexual fruiting-body formation in either haploid or diploid strains carrying an additional incomplete chromosome and hence are disomic or trisomic, respectively, for part of linkage group II. The incomplete chromosome is lost at a frequency of 2 to 3% from disomic and trisomic strains, but surprisingly this loss is not increased in the presence of the haploidizing agent, benlate. A new locus (clyA), whose phenotype is altered colony morphology, is assigned to the region of linkage group II encompassed by the fragment chromosome.

The use of restriction fragment length polymorphisms and DNA duplications to study the organization of the actin multigene family in Dictyostelium discoideum.

The techniques of restriction fragment length polymorphism analysis and examination of gene copy number in duplication-bearing Dictyostelium discoideum strains have been used to map four actin genes of the wild-type strain NC4 to specific linkage groups. In part, this was accomplished by identification of restriction fragments corresponding to particular cloned actin genes using gene-specific probes from unique sequence 5' and 3' untranslated regions. Cloned gene Actin 8 (designation act-8) maps to linkage group I; Actins 12 (act-12) and M6 (actM6) to linkage group II. An uncloned gene (act-100) also maps to linkage group II in the same region as actM6, as defined by a chromosomal duplication. From analysis of other wild isolates of D. discoideum, it was determined that in these isolates at least two actin genes map to linkage group I and at least four map to linkage group II. These results demonstrate the utility of molecular techniques in genetic analysis of Dictyostelium, particularly for developmentally regulated genes that have been cloned but that have no identified mutant phenotypes.

Dictyostelium discoideum nuclear plasmid Ddp5 is a chimera related to the Ddp1 and Ddp2 plasmid families.

The 14,955-bp Dictyostelium discoideum nuclear plasmid Ddp5 contains six transcribed open reading frames. One of these is related to the rep gene of the Ddp2 plasmid, and the other five are related to genes present on the Ddp1 plasmid. The absence of a homolog of the Ddp1 G1 gene, coupled with the presence of the Ddp2 rep gene homolog and of a 1.6-kb inverted repeat analogous to the inverted repeats on members of the Ddp2 plasmid family, suggests that Ddp5 uses Ddp2-like replication and copy number control mechanisms and that it should be assigned to the Ddp2 plasmid family. Ddp5 carries genes homologous to the D1/D3 and D2 genes of the Ddp1 plasmid as well as the Ddp1 G2/G3/D4, G5/D6, and G6/G4/D5 genes. The products of the Ddp5 G2-like, G5-like, and G6-like genes are likely to be transcription factors regulating the expression of themselves and of the other Ddp5 genes. The D1-like and D2-like genes may confer a selective advantage to plasmid-bearing cells, because they can be deleted from plasmid-based shuttle vectors with no apparent effect on vector maintenance. Updated sequence information for the Ddp1 G5/D6, D1/D3, and D2 genes as well as the Dmp1 and Dmp2 G5-like genes is presented. The locations of introns in the G5-like and D1-like genes of Ddp5 and in the homologous genes of the Ddp1, Dmp1, and Dmp2 plasmids were identified. These introns all have GU at the 5' intron border and AG at the 3' intron border, are short (59 to 71 nucleotides), and are AT-rich. A conserved HHCC domain was identified in the G5 proteins; this is a putative zinc binding domain and may be involved in protein-DNA interaction.