Characterization of IS1515, a Functional Insertion Sequence in Streptococcus pneumoniae

We describe the characterization of a new insertion sequence, IS1515, identified in the genome of Streptococcus pneumoniae I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585–602, 1959). A copy of this element located in the cap1E_I41R gene was sequenced. The 871-bp-long IS1515 element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS1381 from S. pneumoniae, ISL2 from Lactobacillus helveticus, IS702 from the cyanobacterium Calothrix sp. strain PCC 7601, and IS112 from Streptomyces albus G. IS1515 appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain I41R is the result of both the presence of an IS1515 copy and a frameshift mutation in the cap1E_I41R gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS1515 behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS1515 were also able to excise and move to another locations in the chromosome of S. pneumoniae. To our knowledge, this is the first report of a functional IS in pneumococcus.     

The IS1167 Insertion Sequence Is a Phylogenetically Informative Marker Among Isolates of Serotype 6B Streptococcus pneumoniae

The phylogenetic utility of the IS1167 insertion sequence was examined with restriction fragment length polymorphism (RFLP) analyses of a sample of 50, predominantly invasive, capsular serotype 6B Streptococcus pneumoniae isolates previously characterized by multilocus enzyme electrophoresis (MLEE). The strains represented a genetically diverse assemblage of 34 distinct clonotypes composed of 26 restriction fragment types and 23 multilocus enzyme types. All isolates carried the IS1167 insertion sequence, with an average of 9.5 copies. The cross-classification of isolates based on RFLP and MLEE typing schemes was 81% concordant. Phylogenetic analyses demonstrated a significant (P < 0.0001) association between strains of a given RFLP lineage with those of a given MLEE lineage. A significant correlation (P < 0.00004) was also found between the proportion of restriction fragments shared by any given pair of isolates and their genetic distances estimated from the MLEE data. Parity between the two genetic markers implied that the sampled isolates were in linkage disequilibrium. The existence of nonrandom associations among genetic loci was confirmed by Monte Carlo analyses of the MLEE data. These studies, thus, demonstrated that invasive pneumococcal isolates of a single capsule type recovered on a regional scale can retain a largely clonal population structure over a period of 8 years. The ability to detect linkage disequilibrium and generate relatively congruent dendrograms based on distance and parsimony methods suggested that the restriction fragment data were robust to phylogenetic analysis. Received: 20 May 1997 / Accepted: 20 November 1997     

Competence in Streptococcus pneumoniae Is a Response to an Increasing Mutational Burden

Competence for genetic transformation in Streptococcus pneumoniae has previously been described as a quorum-sensing trait regulated by a secreted peptide pheromone. Recently we demonstrated that competence is also activated by reduction in the accuracy of protein biosynthesis. We have now investigated whether errors upstream of translation in the form of random genomic mutations can provide a similar stimulus. Here we show that generation of a mutator phenotype in S. pneumoniae through deletions of mutX, hexA or hexB enhanced the expression of competence. Similarly, chemical mutagenesis with the nucleotide analog dPTP promoted development of competence. To investigate the relationship between mutational load and the activation of competence, replicate lineages of the mutX strain were serially passaged under conditions of relaxed selection allowing random accumulation of secondary mutations. Competence increased with propagation in these lineages but not in control lineages having wild-type mutX. Resequencing of these derived strains revealed between 1 and 9 single nucleotide polymorphisms (SNPs) per lineage, which were broadly distributed across the genome and did not involve known regulators of competence. Notably, the frequency of competence development among the sequenced strains correlated significantly with the number of nonsynonymous mutations that had been acquired. Together, these observations provide support for the hypothesis that competence in S. pneumoniae is regulated in response to the accumulated burden of coding mutations in the bacterial genome. In contrast to previously described DNA damage response systems that are activated by physical lesions in the chromosome, this pneumococcal pathway may represent a unique stress response system that monitors the coding integrity of the genome.     

Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor

Background

The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.

Methodology

In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.

Conclusions

Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.     

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcβ1-3Gal, GalNAcβ1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H₂O₂ and CO₂. This mutant produced decreased concentrations of H₂O₂ and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo     

Genetic Characterization and Use of a Restriction Fragment Length Variant in the Hypotrichous Ciliate Euplotes crassus1

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.     

Streptococcus pneumoniae DivIVA: localization and interactions in a MinCD-free context

To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA(SPN)) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in the divIVA null mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putative S. pneumoniae cell division proteins. Finally, a missense divIVA mutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of the divIVA mutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA in S. pneumoniae seems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA(SPN) raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.     

Endogenous DNases as a Tool for Isolation of Nuclear Matrix: Critical Parameters of Nucleolysis

The degree of nucleolysis is of critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences, as demonstrated using an example of inactive (c-fos, c-myc, andC ) and active (p53, albumin, and28S rRNA) genes in resting hepatocytes. The optimal degree of nucleolysis is characterized by degradation of loop domains of chromatin, with the relatively uniform molecular weight distribution of DNA being preserved. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched with or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix     

Conservation as It Is: Illicit Resource Use in a Wildlife Reserve in India

While wildlife conservation efforts have become increasingly aggressive around the world, illicit use of resources in conservation areas has not subsided, raising questions about the ecological character of noncompliance activities. This paper reviews the results of research conducted amongst foresters and households living adjacent to a wildlife sanctuary in India in a period following a ban of human use activities. Using a formal survey supplemented by interviews and participant observation, the research sought to determine the intensity of illicit use, the demographics of resource users, and the attitudes of rulebreakers. The results suggest that noncompliance with conservation restrictions is nearly universal, that forest use is highly specialized, that available village resource assets do little to offset forest use, and that rule-breakers prefer current governance arrangements. These results paint a picture of deeply institutionalized forest use that suggests serious barriers to any simple enforcement solutions or governance reforms.     

Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae

Regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, genes involved in DNA uptake and recombination and a link between these two gene sets. The alternative sigma factor ComX provides this link. ComE, the response regulator of the quorum-sensing system, is required for expression of ComX and other early genes. However, an unknown ComE-dependent regulator is also required for competence when comX is expressed under control of the raffinose-responsive promoter of the aga operon. The gene comW (SP0018) is required for a high level of competence and is regulated by the quorum-sensing system, but its function is unknown. To explore its role further, comW was cloned into the multicopy plasmid pMSP3535, under the control of a nisin-inducible promoter (P(N)), and transformed into pneumococcal strains containing a raffinose-inducible comX gene (P(R)::comX). Further introduction of a comE deletion blocked the endogenous CSP signal transduction pathway. In the resulting strain, competence was independent of CSP but depended on treatment with both nisin and raffinose, showing that coexpression of comW and comX complemented the comE deficiency. ComX protein accumulation and expression of a late competence gene in the above strain support the conclusion that ComW is a new positive factor involved in competence regulation.     

Habitat Use and Activity Patterns as an Indication of Fragment Quality in a Strepsirrhine Primate

Considerable intraspecific variation exists in responses to habitat fragmentation owing to regional differences in fragment quality. Habitat use and activity patterns of resident populations are related to the abundance and predictability of resources in time and space, and thus space use is one of the most important considerations for evaluating habitat quality. Here I examine inter- and intrasite differences in the ranging behavior; use of the matrix, i.e., surrounding nonhabitat; and activity budgets of ring-tailed lemurs (Lemur catta) inhabiting two forest fragments—Anja Special Reserve and Tsaranoro Valley Sacred Forest—in Madagascar’s central highlands to test the prediction that intraspecific variation in habitat use and activity patterns is indicative of fragment quality. At Anja, Lemur catta has access to abundant year-round anthropogenic resources (introduced fruit trees, agricultural crops, water), whereas the population at Tsaranoro relies on seasonally variable and patchily distributed resources throughout the forest and matrix. Lemur catta at Anja occupy smaller home ranges; have shorter daily path lengths (DPLs); and spend less time foraging and more time resting, locomoting, and engaging in social and territorial behavior. In contrast, groups at Tsaranoro occupy larger home ranges, have longer DPLs, rely heavily on the matrix, and expend most of their energy in foraging behavior. These differences point to variation in the relative habitat quality of the two fragments and are thus an important consideration for conservation managers when assessing the capacity of forest fragments to sustain populations.     

Excision-repair capacity in Streptococcus pneumoniae: cloning and expression of a uvr-like gene

Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to UV light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coli. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.     

MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.     

Use of a cloned DNA fragment to analyze the fate of donor DNA in transformation of Streptococcus pneumoniae

The integration of donor label into the recipient fragment is followed during transformation of Streptococcus pneumoniae. The method used involves gel analysis of restriction endonuclease-treated recipient DNA after recombination with a radioactively labeled homologous cloned fragment.