Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells

The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (NpnN) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular NpnN. The normal level of 3 microM adenylated NpnN (ApnN) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular NpnN in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells.     

Plasmid-encoded gamma-hexachlorocyclohexane degradation genes and insertion sequences in Sphingobium francense (ex-Sphingomonas paucimobilis Sp+)

The lin genes encode the gamma-hexachlorocyclohexane (gamma-HCH or lindane) catabolic pathway in lindane-degrading strains. The location and stability of these genes have been explored in the lindane-degrading Sphingobium francense strain Sp+, and in two non-lindane-degrading mutants (Sp1- and Sp2-). The lin genes, linA, linB, linE and linX were localized by hybridization on three of the six plasmids of the S. francense strain Sp+ showing dispersal within the genome. The linC gene was detected by PCR, but was not detected by hybridization on any of the plasmids. The hybridization of the linA and linX genes was negative with the two non-lindane-degrading mutants S. francense strains, Sp1- and Sp2-. The dynamic of this genome associated with gene loss and acquisition, and plasmid rearrangement was explored by a search for associated insertion sequences. A new insertion sequence, ISSppa4, belonging to the IS21 family was detected and compared with IS6100 and ISsp1. Insertion sequence localization was explored on different hybridization patterns (plasmid, total genome) with the lindane-degrading Sp+ strain and the two non-degrading derivatives (Sp1-, Sp2-). Insertion sequence movement and plasmid rearrangement could explain the emergence of the non-lindane-degrading mutants.     

Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.     

Mapping of sex determination loci on the white campion (Silene latifolia) Y chromosome using amplified fragment length polymorphism

S. latifolia is a dioecious plant with morphologically distinct sex chromosomes. To genetically map the sex determination loci on the male-specific Y chromosome, we identified X-ray-induced sex determination mutants that had lost male traits. We used male-specific AFLP markers to characterize the extent of deletions in the Y chromosomes of the mutants. We then compared overlapping deletions to predict the order of the AFLP markers and to locate the mutated sex-determining genes. We found three regions on the Y chromosome where frequent deletions were significantly associated with loss of male traits. One was associated with hermaphroditic mutants. A second was associated with asexual mutants that lack genes needed for early stamen development and a third was associated with asexual mutants that lack genes for late stages of stamen development. Our observations confirmed a classical genetic prediction that S. latifolia has three dispersed male-determining loci on the Y chromosome, one for carpel suppression, one for early stamen development, and another for late stamen development. This AFLP map provides a framework for locating genes on the Y chromosome and for characterizing deletions on the Y chromosomes of potentially interesting mutants.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

Heavy ion mutagenesis: Linear energy transfer effects and genetic linkage

We have characterized a series of 69 independent mutants at the endogenoushprt locus of human TK6 lymphoblasts and over 200 independent S 1-deficient mutants of the humanxhamster hybrid cell line A_L arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/µm). We find that large deletions are common. The entirehprt gene (>44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entirehprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency ofhprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of thehprt locus to mutation is likely due to tight linkage to a gene that is required for viability.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Identification of genes required for growth under ethanol stress using transposon mutagenesis in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.     

Genomic island 2 is an unstable genetic element contributing to Brucella lipopolysaccharide spontaneous smooth-to-rough dissociation

Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages.     

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes.

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.     

Identification of genes relevant to symbiosis and competitiveness in Sinorhizobium meliloti using signature-tagged mutants

Sinorhizobium meliloti enters an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. In order to identify S. meliloti genes required for symbiosis and competitiveness, a method of signature-tagged mutagenesis was used. Two sets, each consisting of 378 signature-tagged mutants with a known transposon insertion site, were used in an experiment in planta. As a result, 67 mutants showing attenuated symbiotic phenotypes were identified, including most of the exo, fix, and nif mutants in the sets. For 38 mutants in genes previously not described to be involved in competitiveness or symbiosis in S. meliloti, attenuated competitiveness phenotypes were tested individually. A large part of these phenotypes was confirmed. Moreover, additional symbiotic defects were observed for mutants in several novel genes such as infection deficiency phenotypes (ilvI and ilvD2 mutants) or delayed nodulation (pyrE, metA, thiC, thiO, and thiD mutants).     

Bacillus subtilis mutants with alterations in ribosomal protein S4.

Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsD1 allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

Contributions of the viral genetic background and a single amino acid substitution in an immunodominant CD8+ T-cell epitope to murine coronavirus neurovirulence

The immunodominant CD8+ T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHM(N514S)), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHM(N514S) was not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHM(N514S)/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.     

The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution

The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans

Spontaneous mutants of Streptococcus mutans GS-5 defective in sucrose-dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS-5 homologous tandem genes, gtfB and gtfC, coding for GTF-I and GTF-S activities respectively, were demonstrated to undergo recombination when introduced into recombination-proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization-defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization-defective mutants in cultures of S. mutans.     

Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV.