Streptococcus cuniculi sp. nov., isolated from the respiratory tract of wild rabbits.

Biochemical and molecular genetic studies were performed on four unknown Gram-stain-positive, catalase-negative, coccus-shaped organisms isolated from tonsils (n = 3) and nasal samples (n = 1) of four wild rabbits. The micro-organism was identified as a streptococcal species based on its cellular morphological and biochemical tests. Comparative 16S rRNA gene sequencing confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any recognized species of this genus. The closest phylogenetic relative of the unknown cocci from wild rabbits was Streptococcus acidominimus NCIMB 702025(T) (97.9% 16S rRNA gene sequence similarity). rpoB and sodA sequence analysis of the novel isolate showed interspecies divergence of 16.2% and 20.3%, respectively, from the type strain of its closest 16S rRNA gene phylogenetic relative, S. acidominimus. The novel bacterial isolate could be distinguished from the type strain of S. acidominimus by several biochemical characteristics, such as the production of esterase C4, acid phosphatase and naphthol-AS-BI-phosphohydrolase and acidification of different sugars. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus cuniculi sp. nov. The type strain is NED12-00049-6B(T) ( = CECT 8498(T) = CCUG 65085(T)).

Analysis of yeast protein kinases using protein chips.

We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.

A genetic comparison of pig, cow and trout isolates of Lactococcus garvieae by PFGE analysis.

AIMS: Genetic comparison of Lactococcus garvieae isolated from mammals and fish. METHODS AND RESULTS: One hundred and ninety-seven L. garvieae isolates obtained from trout (n = 153), cow (n = 7) and pigs (n = 37) were genetically characterized by determining their pulsed-field gel electrophoresis (PFGE) profiles after macrorestriction with Bsp120I. Overall, L. garvieae isolates from pigs, cow and trout exhibited distinct PFGE patterns, with a low genetic relationship between them. Isolates from trout generated two pulsotypes [Genetic diversity (GD) 0.01] showing that the fish isolates were more genetically homogenous than the others. The L. garvieae isolates from cows displayed five (GD 0.71) different pulsotypes, while the swine isolates displayed 13 different pulsotypes (GD 0.35). Twenty-one of the 37 swine strains (56.8%) were grouped in a single cluster that included two closely related (93% similarity) pulsotypes. These pulsotypes exhibited a high frequency of isolation from different organs of the animals, and they were also broadly distributed among herds, suggesting a wide distribution across the swine population. This suggests that L. garvieae might be able to colonize different organs of the swine cardio-respiratory system. CONCLUSIONS: Results indicate that most L. garvieae isolates from pigs and trout exhibited a distinct genetic background. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study describes the isolation of L. garvieae from both diseased and healthy pigs for the first time, and the findings suggest that pigs could be a previously unknown reservoir of this pathogen.

Global analysis of protein activities using proteome chips.

To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast.

BACKGROUND: In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses. RESULTS: We found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK. CONCLUSIONS: Pkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2.

BACKGROUND: Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown. RESULTS: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBalpha, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBalpha to one that phosphorylates both Thr308 and Ser473 of PKBalpha in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBalpha in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies. CONCLUSIONS: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase.

BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

Molecular cloning and characterization of two phosphatase 2A catalytic subunit genes from Arabidopsis thaliana.

The plant Arabidopsis thaliana contains five isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) that can be grouped into two families, one composed by isoforms PP2A-1, -2 and -5 and the other composed by isoforms PP2A-3 and PP2A-4. An Arabidopsis genomic library was screened and several clones corresponding to genes PP2A-3 and PP2A-4 were isolated and analysed. Both genes span over approximately 4.5kbp and are composed of 11 exons and 10 introns that show identical organization. Their untranslated regions are also highly conserved, suggesting that the two genes derive from a common ancestral gene. However, the position of intron/exon junctions completely differs from that of the human PP2A genes. Two transcription start sites have been found in the PP2A-3 gene, the major one mapping at nucleotide position -188 from the translation start codon, whereas only one is observed in PP2A-4 (-145). Functional gene promoter analysis reveals that elements required for transient expression of PP2A-3 and PP2A-4 on a protoplast system are contained within a region of about 600bp upstream from the transcription start sites. This is the first report on the cloning and characterization of genes encoding catalytic subunits of Ser/Thr protein phosphatases 2A in higher plants.

The PPZ protein phosphatases are involved in the maintenance of osmotic stability of yeast cells.

We have recently reported the existence in the yeast Saccharomyces cerevisiae of a gene named PPZ1, encoding a novel Ser/Thr phosphatase characterized by a large, Ser-rich amino-terminal extension, and suggested the existence of a related gene product that could have overlapping functions. We have now amplified by polymerase chain reaction techniques a genomic fragment of about 600 bp corresponding to this second gene (PPZ2). This fragment hybridizes to an mRNA of about the same size as the PPZ1 message but the amount of PPZ2 mRNA peaks at the stationary phase, when almost no PPZ1 mRNA is found. The PPZ2 fragment was interrupted in vitro and used to transform diploid heterozygous ppz1 PPZ2 cells. Haploid cells carrying the double mutation ppz1 ppz2 were unable to grow in the presence of 5 mM caffeine. However, the mutants did survive when osmotically stabilized in the presence of 1 M sorbitol. The evidence obtained suggests that PPZ1 and PPZ2 may be structurally and functionally related and points to an involvement of these phosphatases in functions related to the maintenance of cell integrity.

Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain.

A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1. Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase B alpha (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides.

Streptococcus plurextorum sp. nov., isolated from pigs.

Biochemical and molecular genetic studies were performed on an unknown Gram-positive, catalase-negative, coccus-shaped organism isolated from clinical samples from pigs. On the basis of the results of cellular morphological and biochemical tests, the organism was identified as a streptococcal species. 16S rRNA gene sequence comparisons confirmed its identification as a member of the genus Streptococcus, but the organism was distinct from any recognized species of this genus. The closest phylogenetic relative of the unknown organism corresponded to Streptococcus suis NCTC 10234(T) (97.2 % 16S rRNA gene sequence similarity) and this phylogenetic position was confirmed by analysis of rpoB and sodA sequences. DNA-DNA hybridization studies showed that the unidentified organism produced a DNA reassociation value of 36.6 % with respect to S. suis NCTC 10234(T). The novel bacterium was distinguished from S. suis and other Streptococcus species using biochemical tests. On the basis of phenotypic and phylogenetic data, the unidentified organism represents a novel species of the genus Streptococcus, for which the name Streptococcus plurextorum sp. nov. is proposed. The type strain is 1956-02(T) (=CECT 7308(T)=CCUG 52972(T)).

Aerococcus suis sp. nov., isolated from clinical specimens from swine.

Biochemical and molecular genetic studies were performed for five isolates of unknown Gram-positive, catalase-negative, cocci-shaped micro-organisms obtained from clinical samples from pigs. The micro-organisms were tentatively identified as Aerococcus species on the basis of the results from cellular morphological and biochemical tests. 16S rRNA gene sequencing studies confirmed the provisional identification of the isolates as members of the genus Aerococcus, but the micro-organism did not correspond to any recognized species of this genus. The nearest phylogenetic relatives of these unknown cocci isolated from pigs were Aerococcus viridans (95.9 % 16S rRNA gene sequence similarity) and Aerococcus urinaeequi (95.8 %). The unknown bacterium, however, was distinguishable from these two species and from other animal aerococci by using biochemical tests. On the basis of both phenotypic and phylogenetic findings, the isolates represent a novel species of the genus Aerococcus, for which the name Aerococcus suis sp. nov. is proposed. The type strain is 1821/02(T) (=CECT 7139(T)=CCUG 52530(T)).

Molecular cloning and analysis of a yeast protein phosphatase with an unusual amino-terminal region.

DNA fragments containing structural characteristics found in Ser/Thr protein phosphatases were amplified by polymerase chain reaction from yeast genome. Amplification was carried out by using degenerate oligonucleotides encoding conserved sequences found in type 1, 2A, and 2B phosphatases. A 215-base pair amplification fragment was used to screen a size-selected library, and a positive clone was isolated and sequenced. Nucleotide sequencing revealed a 2076-base pair open reading frame encoding a 692-amino acid protein. The carboxyl half of the protein is structurally related to type 1 phosphatases and virtually identical with the sequence reported as PPZ1, whereas the amino-terminal half of the protein is unrelated to sequences found in other protein phosphatases. This region is very rich in Ser residues and presents a high number of basic amino acids. Therefore, the gene product, on the basis of its unique structure, would represent a novel class of protein phosphatase. The gene, which is located at chromosome XIII, is transcribed as a mRNA of about 2.7 kilobases, and the amount of message has been found to increase 3- to 4-fold during the culture. The product of the gene PPZ1 was identified by immunoblot analysis of cell extracts as a 75-kDa protein, and the amount of immunoreactive protein was increased in cells carrying multiple copies of the gene. Disruption of the gene resulted in viable cells, with no detectable phenotypic change, indicating that the gene is not essential for growth.

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo.

3-phosphoinositide-dependent protein kinase-1 (PDK1) expressed in unstimulated 293 cells was phosphorylated at Ser-25, Ser-241, Ser-393, Ser-396 and Ser-410 and the level of phosphorylation of each site was unaffected by stimulation with insulin-like growth factor-1. Mutation of Ser-241 to Ala abolished PDK1 activity, whereas mutation of the other phosphorylation sites individually to Ala did not affect PDK1 activity. Ser-241, unlike the other phosphorylation sites on PDK1, was resistant to dephosphorylation by protein phosphatase 2A(1). Ser-241 lies in the activation loop of the PDK1 kinase domain between subdomains VII and VIII in the equivalent position to the site that PDK1 phosphorylates on its protein kinase substrates. PDK1 expressed in bacteria was active and phosphorylated at Ser-241, suggesting that PDK1 can phosphorylate itself at this site, leading to its own activation.

The gene DIS2S1 is essential in Saccharomyces cerevisiae and is involved in glycogen phosphorylase activation.

S. cerevisiae gene DIS2S1, which codes for a protein very similar to the catalytic subunit of mammalian protein phosphatase 1, was disrupted "in vitro". Diploid yeast cells were transformed and sporulated. Tetrad analysis demonstrated that disruption of DIS2S1 is lethal for the cell. Glycogen phosphorylase alpha and glycogen synthase activity ratio were measured in diploids carrying a disrupted allele of the gene. Phosphorylase was dramatically activated in mutant cells but, under the same conditions, glycogen synthase activity was essentially identical in both mutant and wild-type cells.

Identification and molecular cloning of two homologues of protein phosphatase X from Arabidopsis thaliana.

In a recent paper [Ariño et al., Plant Mol Biol 21: 475-485 (1993)] we reported the amplification of a DNA fragment (AP-2) from the genome of Arabidopsis thaliana encoding an amino acid sequence corresponding to a Ser/Thr protein phosphatase distantly related to type 2A protein phosphatases. In this paper we report the use of the AP-2 fragment to isolate several cDNA clones from a leaf cDNA library. Two of these (EP124 and EP129) largely overlap and contain the AP-2 sequence, whereas a third clone (EP128) is different although very related in sequence (86% of identity). Clones EP124/EP129 and EP128 were found to encode two highly related polypeptides (93% identity) of 305 residues, showing a very high identity (83%) to the catalytic subunit of protein phosphatase X (PPX) from rabbit. Therefore, they have been named PPX-1 (EP124/EP129) and PPX-2 (EP128). Southern blot analysis of genomic DNA indicates that only these two genes encoding phosphatases closely related to PPX are present in the genome of A. thaliana. Both PPX-1 and PPX-2 are expressed at very low levels in A. thaliana flowers, leaves, stems and roots. The expression levels of four previously identified type 2A phosphatases are higher than those of PPX genes. PP2A-1 appears to be the major mRNA species detected in all the tissues analyzed.

The gene PPG encodes a novel yeast protein phosphatase involved in glycogen accumulation.

Degenerate oligonucleotides were used to selectively amplify yeast genomic sequences related to Ser/Thr protein phosphatases. Among the sequences obtained, clone ST4-2 was found to code for a novel sequence related to previously known phosphatases. A size-selected yeast genomic library was constructed and screened using clone ST4-2 as probe, and one positive clone, named PPG, was isolated. DNA sequencing of a 1.8-kilobase pair fragment of this clone revealed an open reading frame of 1104 base pairs which codes for a 368-amino acid protein. On the basis of its amino acid sequence, the product of gene PPG would be an acidic protein, structurally more related to type 2A than to type 1 or 2B phosphatases, and is characterized by an extension of about 50 amino acids at the carboxyl terminus. The gene, which is located in chromosome XIV, is expressed as a 1.3-kilobase mRNA and is not essential for growth. Haploid mutants carrying a disrupted copy of the gene were able to grow in glucose as well as in other carbon sources, but they accumulated less glycogen than the wild type strain. However, the state of activation of glycogen synthase was essentially identical in wild type and mutant cells. The finding that, in early exponential phase, mutant cells contain higher levels of glycogen phosphorylase a, in addition to a lower amount of total glycogen synthase activity observed in medium-late exponential phase, could account for the difference found in glycogen accumulation.