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.

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.

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.

Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review.

Many strains of Streptococcus thermophilus synthesize extracellular polysaccharides. These molecules may be produced as capsules that are tightly associated with the cell, or they may be liberated into the medium as a loose slime (i.e., "ropy" polysaccharide). Although the presence of exopolysaccharide does not confer any obvious advantage to growth or survival of S. thermophilus in milk, in situ production by this species or other dairy lactic acid bacteria typically imparts a desirable "ropy" or viscous texture to fermented milk products. Recent work has also shown that exopolysaccharide-producing S. thermophilus can enhance the functional properties of Mozzarella cheese, but they are not phage-proof. As our understanding of the genetics, physiology, and functionality of bacterial exopolysaccharides continues to improve, novel applications for polysaccharides and polysaccharide-producing cultures are likely to emerge inside and outside the dairy industry. This article provides an overview of biochemistry, genetics, and applications of exopolysaccharide production in S. thermophilus.

Mechanism of action of the Rep protein from the Dictyostelium Ddp2 plasmid family.

The two-hybrid system was used to show that the Rep proteins from three members of the Dictyostelium discoideum Ddp2 plasmid family, Ddp2, Ddp5, and Ddp6, form homomultimers but not heteromultimers when expressed in yeast cells. The results with deletion mutations suggest that multiple regions of the Rep proteins are involved in the multimerization. Electrophoretic mobility shift assays with heterologously expressed and purified Ddp2 Rep protein showed that it is a DNA binding protein. The nucleosomal organization of Ddp2 and Ddp6 in their inverted repeat and promoter regions was investigated. Analysis of mutants derived from the Ddp6 plasmid revealed that its Rep protein is required for nucleosome positioning (i.e., phasing) to occur in the promoter region. On the other hand, nucleosome positioning in the inverted repeat regions of both plasmids is not dependent on Rep protein but on either a feature of the DNA sequence or the binding of cellular factors, perhaps the Dictyostelium origin recognition complex. Rep protein is likely involved in transcription regulation and control of DNA replication, specifically amplification of plasmid at low copy numbers. The formation of homomultimers may be required for their regulatory activity.

Dgp1 and Dfp1 are closely related plasmids in the Dictyostelium Ddp2 plasmid family.

Dictyostelium plasmids Dgp1 and Dfp1, two members of the Ddp2 plasmid family, are 86% identical in nucleotide sequence. These small (4481 and 5015 bp), high copy number, nuclear plasmids carry both a gene homologous to the Ddp2 rep gene and a long 0.47- to 0. 48-kb inverted repeat region. Their Rep proteins are 82.8% identical in amino acid sequence and carry all 10 of the conserved peptide sequence motifs found in the Ddp2 family Rep proteins. Unlike other members of this family, Dgp1 carries two copies and Dfp1 carries four copies of a 162- to 166-bp direct repeat element. Both the direct and inverted repeat elements, as well as the promoter of the rep gene, are highly conserved (81 to 90% identical) between Dgp1 and Dfp1. In contrast, these regions are not highly conserved and the Rep proteins are only about 40% identical among the other known members of the plasmid family.

Dictyostelium discoideum nuclear plasmid Ddp6 is a new member of the Ddp2 plasmid family.

Ddp6 is a high-copy number, circular plasmid found in the nucleus of the simple eukaryote Dictyostelium discoideum wild isolate NC47.2. The complete nucleotide sequence, 5257 bp, shows that Ddp6 has a structure similar to that of other members of the Ddp2 plasmid family: a single long 2.8-kb open reading frame (rep gene) and an inverted repeat containing a pair of 654-bp elements. A single constitutively expressed 3.3-kb transcript of the rep gene was detected in RNA prepared from vegetative and developmental cells. Maintenance assays revealed that sequences within the inverted repeat and the intact Ddp6 ORF are essential for maintenance of the plasmid. Mutation of the inverted repeat, or of the rep gene, lowered the plasmid copy number but did not affect autonomous replication of shuttle-vector constructs. Comparisons of the predicted protein products of the rep genes of five members of the Ddp2 plasmid family identified a set of ten conserved features distributed throughout the peptides.

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.

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification.

Wild-type Dictyostelium discoideum cells growing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2-3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.

The replication origin position and its relationship to a negative trans-acting transcription regulator encoded by Dictyostelium discoideum nuclear plasmid Ddp1.

The replication origin of the Dictyostelium discoideum plasmid Ddp1 was localized to a 543-bp region. This includes most of the AT-rich intergenic region between the G1 and G5/D6 genes containing both of their promoters and multiple copies of a TTTTGACT repeat. The G5/D6 gene, which lies adjacent to, and partially overlaps, the 543-bp origin region, encodes a trans-acting factor that negatively regulates transcription of the G4/D5 gene. Inactivation of the G5/D6 gene led to expression of a transcript (G6) 0.2 kb larger than the D5 transcript from the G4/D5 gene in vegetative and developing cells. The G5/D6 gene also regulates transcription of the G1, G2/G3/D4 and G5/D6 genes either alone or in concert with other Ddp1 gene products.

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.

Compatible Dictyostelium mucoroides nuclear plasmids Dmp1 and Dmp2 both belong to the Ddp1 plasmid family.

Dictyostelium mucoroides plasmids Dmp1 and Dmp2 are naturally occurring compatible members of the Dictyostelium Ddp1 plasmid family found in the same wild isolate strain. The nucleotide sequences of Dmp1 (5983 bp) and Dmp2 (6018 bp) are 74% identical and each carries open reading frames (ORFs) similar to the G1 and G5 ORFs of the Dictyostelium discoideum plasmid Ddp1. The predicted protein product of the Ddp1 G1 ORF is 49% similar to that of the Dmp1 G1-like ORF and 52% similar to that of the Dmp2 G1-like ORF. For the G5 and G5-like ORFs the corresponding values are 47 and 43%, respectively. The G1 and G5 ORFs of Ddp1 are transcribed during both vegetative growth and development of the asexual fruiting body. The G1-like ORF of Dmp2 is expressed in vegetative and developing cells, while that of Dmp1 appears to be expressed mostly in developing cells. The G5-like ORFs of Dmp1 and Dmp2 are expressed in both vegetative and developing cells. Dmp1 and Dmp2 differ from Ddp1 in (1) lacking homologs for the Ddp1 G2/G3/D4, G4/D5, D1/D3, and D2 ORFs; (2) containing multiple copies of a 173 bp direct repeat; and (3) having a different orientation of the G1 ORF relative to the G5 ORF. These findings suggest that the basic replicon unit of the Ddp1 plasmid family is composed of an origin of replication coupled to G1-like and G5-like genes. The additional ORFs and direct repeat elements in Ddp1, Dmp1, and Dmp2 may provide accessory functions beneficial to plasmid maintenance. Shuttle vectors based on Dmp1 or Dmp2 replicate in D. discoideum transformants.

Nucleotide sequence of Ddp1, a high copy number nuclear plasmid of Dictyostelium discoideum.

The complete nucleotide sequence of Ddp1, a high copy number 13.7-kbp endogenous nuclear plasmid of Dictyostelium discoideum is presented. Previous studies have shown nine transcripts which map to five different regions of Ddp1, suggesting alternative transcription initiation sites and/or post-transcriptional processing. The sequence presented here shows five long open reading frames corresponding to previously known transcribed regions, as well as an additional reading frame, in the region of the presumed origin of replication. Two of the predicted proteins from the Ddp1 reading frames have potential leader sequences and transmembrane domains, while the rest, if translated, would encode soluble proteins. One of these has both leucine zipper and zinc finger-like motifs. Sequences upstream of the reading frames show strong similarities to chromosomal promoter elements, suggesting that transcription of at least some of the plasmid-encoded genes is regulated by factors which regulate chromosomal transcription in this organism. The region containing the presumptive origin of replication contains the promoters of the major growth-specific gene, as well as a second gene; thus, the processes of transcription and replication of Ddp1 may be intimately linked. The origin-encompassing region has a long homopurine/homopyrimidine stretch, which is similar to sequences shown in vitro to be capable of forming a triple helix structure through Hoogsteen base pairing. Possible roles for these sequences and the products of the ORFs in plasmid maintenance are discussed.

Small circular plasmids of the eukaryote Dictyostelium purpureum define two novel plasmid families.

Two novel groups of circular, nuclear plasmids were discovered in the simple eukaryote Dictyostelium purpureum. They define two new Dictyostelium plasmid families each containing three members: Dpp1A, Dpp1B, and Dpp2 in the Dpp1 family, and Dpp3, Dpp4, and Dpp5 in the Dpp3 family. These plasmids are among the smallest known, ranging in size from 1309 bp (Dpp1A and Dpp1B) to 1961 bp (Dpp4). Family members are very similar. The most distantly related members of the Dpp1 family (Dpp1A and Dpp2) are 89% identical, while the most distantly related members of the Dpp3 family (Dpp3 and Dpp4) are 91% identical. No sequence similarity is found between these plasmid families, or to any other known plasmid or chromosomal DNA sequence. A 72-bp inverted repeat present in one copy in Dpp1A and Dpp1B is tandemly repeated in Dpp2. The Dpp3 family contains a region of 102-160 nucleotides rich in short, overlapping direct sequence repeats. This region is present once in Dpp3 and Dpp5 and is tandemly repeated in Dpp4. The repeat structures in the Dpp1 and Dpp3 families are relatively rich in GC base pairs (29-41%) in comparison to the unique sequence regions of the plasmids (16-22%). The longest open reading frame (ORF) beginning with an AUG codon in these plasmids is 168 bp in the Dpp3 family, although longer ORFs without AUG start codons (up to 201 bp) also exist. Northern blot analysis did not detect any plasmid-specific transcripts in total RNA prepared from vegetative cells carrying Dpp1A or Dpp3.

A G-protein beta-subunit is essential for Dictyostelium development.

Recent studies have demonstrated that G-protein-linked signal transduction pathways play a significant role in the developmental program of the simple eukaryotic organism Dictyostelium. We have reported previously the isolation of a G-protein beta-subunit and present here a more complete analysis of this gene. Low-stringency Southern blots and RFLP mapping studies suggest that the beta-subunit is a unique gene found on linkage group II. Its deduced amino acid sequence of 347 residues is approximately 60% identical to those of the human, Drosophila, and Caenorhabditis elegans beta-subunits. The carboxy-terminal 300 residues are about 70% identical; the amino-terminal 50 residues are quite divergent, containing only 10 identities. At all stages of growth and development, a single 1.9-kb beta-subunit mRNA is present at a high level, and a specific antibody detects a single 37-kD protein. We propose that G-protein heterotrimers are formed when this beta-subunit couples with each of the eight distinct G-protein alpha-subunits that are transiently expressed during development. Targeted disruption of the beta-subunit gene had no effect on the viability of haploid cells, but resulted in the inability of cells to aggregate.

Isolation and characterization of a linear plasmid from the entomopathogenic fungus Ascosphaera apis.

An extrachromosomal DNA plasmid was isolated from both mating types of the entomopathogenic fungus Ascosphaera apis and named pAaL. The subculture in which pAaL was first identified originated from mummified honey bee larvae from an apiary in Wyoming. Very similar, homologous plasmids were found in 9 out of 10 of isolates collected from diverse geographic locations. The plasmid is found inside the mitochondria, has the same buoyant density as mitochondrial DNA in bisbenzamide--CsCl gradients, and does not contain sequences homologous to either mitochondrial DNA or genomic DNA. The plasmid is linear, double-stranded, of 12 kilobase pair, and has a higher copy number than the mitochondrial DNA. Endonuclease and exonuclease digestions suggest that an inverted repeat is probably present at each terminus and that pAaL has two blocked 5' ends, probably due to the presence of terminal binding proteins. Restriction site data showed pAaL to be AT-rich. There were no apparent differences in the growth rate, culture appearance, and reproductive cycle of plasmid-bearing or plasmid-free A. apis isolates. pAaL was stably inherited in the plasmid-free strains, but it was lost in the progeny of crosses and reciprocal crosses between the plasmid-free strain and plasmid-bearing strains. The biological function of this plasmid has not yet been determined.

Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents.

We describe 22 new mutants of D. discoideum that are sensitive to DNA damage. These mutants were isolated on the basis of sensitivity to either temperature, gamma-rays, or 4-nitroquinolone-1-oxide (4NQO). The doses of gamma-rays, ultraviolet light (UV), and 4NQO required to reduce the survival of colony-forming ability of these mutants to 10% (D10) range from 2% to 100% of the D10s for the nonmutant, parent strains. For most of the mutants, those which are very sensitive to one agent are very sensitive to all agents tested and those which are moderately sensitive to one agent, are moderately sensitive to all agents tested. One mutant is sensitive only to 4NQO. Linkage relationships have been examined for 13 of these mutants. This linkage information was used to design complementation tests to determine allelism with previously characterized complementation groups affecting sensitivity to radiation. 4 of the new mutants fall within previously identified complementation groups and 3 new complementation groups have been identified (radJ, radK and radL). Other new loci probably also exist among these new mutants. This brings the number of characterized mutants of D. discoideum which are sensitive to DNA-damaging agents to 33 and the number of assigned complementation groups to 11.

Dictyostelium giganteum plasmid Dgp1 is a member of the Ddp2 plasmid family.

Dgp1, a circular 4.4-kb plasmid found in the nuclei of Dictyostelium giganteum strain DG61, is a member of the same plasmid family as plasmids Ddp2 and pDG1. Dgp1 has sequence similarity to a conserved region of the Ddp2 and pDG1 open reading frames. As with Ddp2 and pDG1, a single large RNA is transcribed from Dgp1. This 3.3-kb transcript is present at about 350 copies per vegetative cell. The transcript abundance decreased about 10-fold in early aggregation and continued at this lower level until late culmination when it returned to the level seen in vegetative cells. Dgp1 has a repeat of several hundred base pairs in a location, relative to the transcribed region, similar to the inverted repeats found in Ddp2 and pDG1. Dgp1 cannot be maintained as a plasmid in Dictyostelium discoideum AX4 cells, suggesting that Dgp1 carries species-specific maintenance elements.