The High Biofilm-Encoding <Emphasis Type="Italic">Bee</Emphasis> Locus: A Second Pilus Gene Cluster in <Emphasis Type="Italic">Enterococcus</Emphasis> <Emphasis Type="Italic">faecalis</Emphasis>?

An Enterococcus faecalis mutant strain with a reduced ability for biofilm formation and primary attachment when compared to the high biofilm-forming wild-type strain was characterized by molecular biological and proteomic approaches. A point mutation in the srt-1 gene, which encodes a sortase-type enzyme and is part of the recently described bee (biofilm enhancer in Enterococcus) gene cluster, could be identified in the mutant strain. The Srt-1 deficiency resulted in a loss of the Bee-2 protein within a high molecular weight complex in cell surface protein extracts, as determined by mass spectrometry. These findings strongly suggest a specific linkage of Bee-2 to Bee-1 and Bee-3 within a complex by Srt-1. Furthermore, the identification of specific pilin motifs conserved in surface proteins of gram-positive bacteria indicated a possible involvement of the bee genes in the formation of pili structures, and may thus play a role in enhancing biofilm formation in Enterococcus faecalis.     

Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK

Short-patch repair of DNA single-strand breaks and gaps (SSB) is coordinated by XRCC1, a scaffold protein that recruits the DNA polymerase and DNA ligase required for filling and sealing the damaged strand. XRCC1 can also recruit end-processing enzymes, such as PNK (polynucleotide kinase 3′-phosphatase), Aprataxin and APLF (aprataxin/PNK-like factor), which ensure the availability of a free 3′-hydroxyl on one side of the gap, and a 5′-phosphate group on the other, for the polymerase and ligase reactions respectively. PNK binds to a phosphorylated segment of XRCC1 (between its two C-terminal BRCT domains) via its Forkhead-associated (FHA) domain. We show here, contrary to previous studies, that the FHA domain of PNK binds specifically, and with high affinity to a multiply phosphorylated motif in XRCC1 containing a pSer-pThr dipeptide, and forms a 2:1 PNK:XRCC1 complex. The high-resolution crystal structure of a PNK–FHA–XRCC1 phosphopeptide complex reveals the basis for this unusual bis-phosphopeptide recognition, which is probably a common feature of the known XRCC1-associating end-processing enzymes.     

Positive and negative selection in murine ultraconserved noncoding elements

There are many more selectively constrained noncoding than coding nucleotides in the mammalian genome, but most mammalian noncoding DNA is subject to weak selection, on average. One of the most striking discoveries to have emerged from comparisons among mammalian genomes is the hundreds of noncoding elements of more than 200 bp in length that show absolute conservation among mammalian orders. These elements represent the tip of the iceberg of a much larger class of conserved noncoding elements (CNEs). Much evidence suggests that CNEs are selectively constrained and not mutational cold-spots, and there is evidence that some CNEs play a role in the regulation of development. Here, we quantify negative and positive selection acting in murine CNEs by analyzing within-species nucleotide variation and between-species divergence of CNEs that we identified using a phylogenetically independent comparison. The distribution of fitness effects of new mutations in CNEs, inferred from within-species polymorphism, suggests that CNEs receive a higher number of strongly selected deleterious mutations and many fewer nearly neutral mutations than amino acid sites of protein-coding genes or regulatory elements close to genes. However, we also show that CNEs experience a far higher proportion of adaptive substitutions than any known category of genomic sites in murids. The absolute rate of adaptation of CNEs is similar to that of amino acid sites of proteins. This result suggests that there is widespread adaptation in mammalian conserved noncoding DNA elements, some of which have been implicated in the regulation of crucially important processes, including development.     

Rhinovirus infection induces extracellular matrix protein deposition in asthmatic and nonasthmatic airway smooth muscle cells

Airway remodeling, which includes increases in the extracellular matrix (ECM), is a characteristic feature of asthma and is correlated to disease severity. Rhinovirus (RV) infections are associated with increased risk of asthma development in young children and are the most common cause of asthma exacerbations. We examined whether viral infections can increase ECM deposition and whether this increased ECM modulates cell proliferation and migration. RV infection of nonasthmatic airway smooth muscle (ASM) cells significantly increased the deposition of fibronectin (40% increase, n = 12) and perlecan (80% increase, n = 14), while infection of asthmatic ASM cells significantly increased fibronectin (75% increase, n = 9) and collagen IV (15% increase, n = 9). We then treated the ASM cells with the Toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid, imiquimod, and pure RV RNA and were able to show that the mechanism through which RV induced ECM deposition was via the activation of TLR3 and TLR7/8. Finally, we assessed whether the virus-induced ECM was bioactive by measuring the amount of migration and proliferation of virus-naive cells that seeded onto the ECM. Basically, ECM from asthmatic ASM cells induced twofold greater migration of virus-naive ASM cells than ECM from nonasthmatic ASM cells, and these rates of migration were further increased on RV-modulated ECM. Increased migration on the RV-modulated ECM was not due to increased cell proliferation, as RV-modulated ECM decreased the proliferation of virus-naive cells. Our results suggest that viruses may contribute to airway remodeling through increased ECM deposition, which in turn may contribute to increased ASM mass via increased cell migration.     

Genetic studies of the Macushi and Wapishana Indians

Summary Blood samples from 509 Macushi and 623 Wapishana Amerindians of Northern Brazil and Southern Guyana have been analyzed with reference to the occurrence of rare variants and genetic polymorphisms of the following 25 systems: (i) Erythrocyte enzymes: acid phosphatase-1, adenosine deaminase, adenylate kinase-k, carbonic anhydrase-1, carbonic anhydrase-2, esterase A_1,2,3, esterase D, galactose-1-phosphate uridyltransferase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, nucleoside phosphorylase, peptidase A, peptidase B, phosphoglucomutase 1, phosphoglucomutase 2, phosphogluconate dehydrogenase, phosphohexoseisomerase, triosephosphate isomerase and (ii) Serum proteins: albumin, ceruloplasmin, haptoglobin, hemoglobin A, hemoglobin A₂ and transferrin. Fifteen different rare variants were detected, involving 11 of these systems. In addition, a previously undescribed variant of ESA_1,2,3 which achieves polymorphic proportions in both these tribes is described. Excluding this variant, the frequency of rare variants is 1.1/1000 in 12510 determinations in the Macushi and 4.7/1000 in 15 396 determinations in the Wapishana. The ESA_1,2,3, polymorphism was not observed in 382 Makiritare, 232 Yanomama, 146 Piaroa, 404 Cayapo, 190 Kraho and 112 Moro. Irregularities in the intratribal distribution of this polymorphism in the Macushi and Wapishana render a decision as to the tribe of origin impossible at present. Gene frequencies are also given for previosly described polymorphisms of 5 systems: haptoglobin, phosphoglucomutase 1, erythrocyte acid phosphatase, esterase D, and galactose-1-phosphate-uridyl-transferase.Research supported by the National Science Foundation and the Energy Research and Development Administration.     

Effect of Drying Medium on Residual Moisture Content and Viability of Freeze-Dried Lactic Acid Bacteria

The effect of various substances on the relationship between residual moisture content and the viability of freeze-dried lactic acid bacteria has been studied. Compounds such as polymers, which display considerable ability in displacing water, showed no protective action during freeze-drying. Adonitol, on the other hand, produced the smallest change in water content at various times during drying and allowed the highest rate of survival.     

Changes in DNA supertwist as a response of Bacillus subtilis towards different kinds of stress

Abstract The silent parD ( kis/kid ) stability operon of plasmid R1 is normally repressed by the co-ordinated action of the Kis and Kid proteins. In this report it is shown that a mutation in repA , the gene of the plasmid replication protein, that reduces two-fold the copy number of the plasmid, leads to the derepression of the parD system. This derepression can be prevented by a suppressor mutation in copB, a copy number control gene of plasmid R1, that increases the efficiency of replication of the repA mutant. Derepression of the wild-type parD system leads to high plasmid stability. These data show the activation of a plasmid stability operon by a mutation that reduces the efficiency of wild-type plasmid replication.     

The cell wall of the human pathogen Candida glabrata: differential incorporation of novel adhesin-like wall proteins

The cell wall of the human pathogen Candida glabrata governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of C. glabrata using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that C. glabrata walls incorporate 50% more protein than Saccharomyces cerevisiae walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that C. glabrata walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-β-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-β-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi, C. glabrata contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like EPA family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of C. glabrata contributes to its adaptability and virulence.     

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.