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.

Comparative genomics of the lactic acid bacteria.

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

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.

Impairments in immune cell function in B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) is a common clonal B cell leukemia that is often accompanied by a multitude of immune abnormalities. Each immune defect may be linked to several of the common complications affecting B-CLL patients. Furthermore, the combined abnormalities constitute a significant immunodeficiency for each patient. Importantly, some of the immune dysfunctions are potentially very relevant to the in vivo survival status of the leukemic B cell. The elucidation of these abnormalities in the circulating non-malignant immune cells of B-CLL patients has generated important insights into the biology of the disease. This discussion reviews the immune abnormalities of the clonal malignant B cells, the polyclonal B cells, and the immunoregulatory T cells and natural killer cells in B-CLL. In addition, we indicate the potential for immunotherapeutic protocols as innovations in treating this disease.

Adenovirus infection of the renal allograft with sparing of pancreas graft function in the recipient of a combined kidney-pancreas transplant.

We report a case of adenovirus infection of the renal allograft in a combined kidney/pancreas transplant recipient. The clinical presentation was renal allograft failure, which eventually reversed. The pancreatic graft function remained stable. A renal biopsy showed massive tubular necrosis associated with a prominent granulomatous reaction. The process had a striking regional distribution within the kidney with the injury and inflammation limited to the outer medulla. Adenovirus type 11 was isolated from renal tissue by culture, and adenovirus was demonstrated by immunofluorescence and electron microscopy in the kidney biopsy. Immunosuppression may result in unusual patterns of response to infectious agents. This case demonstrated tropism of the adenovirus to only selected tubules within the kidney, with sparing of other organ function including, specifically, the pancreas allograft. The differential diagnosis of a granulomatous reaction in the transplant kidney must be expanded to include viral infection, in particular, adenovirus.

Fabrication and characterization of single-mode electro-optic polymer optical fiber.

We report on what we believe is the first demonstration of single-mode polymer optical fiber with embedded electrodes. We show that the electrodes can be used to pole the dye-doped core and to electro-optically phase modulate the light in the waveguide.

Suppressing vibrations in a sheet with a Fabry-Perot photomechanical device.

We report what we believe to be the first demonstration that the vibration amplitude in a stretched plastic sheet can be reduced by as much as 10 dB with a monolithic all-optical mesoscale photomechanical device.

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.

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.

Performance and nutrient digestibility in weanling pigs as influenced by yeast culture additions to starter diets containing dried whey or one of two fiber sources.

Three experiments were conducted using crossbred weanling pigs (7.2 to 8.6 kg; 25 to 29 d of age) to determine the effect on performance and nutrient digestibility of .75% yeast culture (YC) additions to starter diets containing whey or one of two fiber sources. An 18% CP corn-soybean meal basal diet was used in all experiments. In Exp. 1 (n = 192), the addition of YC did not affect ADG, ADFI, or gain: feed ratios (G:F) of pigs fed diets without or with 15% dried whey in two 5-wk trials. In Exp. 2 (n = 174), ADG and ADFI were not affected by YC addition to diets containing no added fiber, 8% soybean hulls (SH), or 8% peanut hulls (PH) in two 5-wk trials. The addition of SH or PH did not affect ADG or ADFI; however, a YC x SH interaction (P < .05) and a YC x PH interaction (P < .10) for G:F indicated that the addition of SH or PH to the diet in the absence of YC reduced G:F, but in the presence of YC, G:F were maintained. In a 3-wk grower phase of one trial in Exp. 2 (n = 54), SH and PH additions decreased ADG (P < .005), whereas YC additions improved ADG (P < .01), particularly for pigs fed diets that also contained SH (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.