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PubMed1998

Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria.

Physiological differentiation (including antibiotic production) in microorganisms usually starts when cells encounter adverse environmental conditions and is frequently accompanied by an increase in the accumulation of intracellular ppGpp. We have found that the acquisition of certain streptomycin-resistant (str) mutations enables cells to overproduce antibiotics, demonstrating an increase in productivity 5- to 50-fold greater than that of wild-type strains. The frequency of such antibiotic-overproducing strains among the str mutants was shown to range from 3 to 46%, as examined with several strains of the genera Streptomyces, Bacillus, and Pseudomonas. Analysis of str mutants from Bacillus subtilis Marburg 168 revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12, changing Lys-56 (corresponding to Lys-43 in Escherichia coli) to Asn, Arg, Thr, or Gln. Antibiotic productivity increased in a hierarchical manner depending upon which amino acid residue replaced Lys at this position. The strA1 mutation, a genetic marker frequently used for mapping, had no effect on antibiotic productivity even though it was found to result in an amino acid alteration of Lys-56 to Ile. Gene replacement experiments with the str alleles demonstrated unambiguously that the str mutation is responsible for the antibiotic overproductivity observed. These results offer a rational approach for improving the production of antibiotic (secondary metabolism) from microorganisms.

Alleles↗
PubMed2000

Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638.

In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyA gene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.

Amino Acid Sequence↗
PubMed2005

The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance.

Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.

Adult↗
PubMed2005

Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system.

The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions.

ATP-Binding Cassette Transporters↗
PubMed1988

Isolation and properties of minB, a complex genetic locus involved in correct placement of the division site in Escherichia coli.

Mutation of Escherichia coli minicell locus (minB) results in aberrant placement of the division septum. In this paper we report the isolation and characterization of the minB locus. Replacement of the chromosomal minB+ allele by cloned minB sequences containing transposon insertions resulted in the minicell phenotype, indicating that minB+ function is required to maintain the normal division pattern. Paradoxically, overexpression of the locus also resulted in the minicell phenotype. The locus codes for several peptides whose expression is coordinately affected by transposon mutations that also eliminate minB+ function. A subset of the minB peptides is sufficient to prevent minicell formation in minB1 mutants or to induce minicell formation when overproduced in wild-type strains, implicating these peptides in the normal process of localization of the division site. The results indicate that minB is a complex locus whose expression must be maintained within certain limits to maintain the normal pattern of localization of the division septum.

Alleles↗
PubMed1991

A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion.

A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene. Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo. Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts. Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts. This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E. coli were attributable to the presence of the overlapping transcript. Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement. This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.

Alleles↗
PubMed1993

Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion.

Pseudomonas aeruginosa secretes elastase in a multistep process which begins with the synthesis of a preproelastase (53.6 kDa) encoded by lasB, is followed by processing to proelastase (51 kDa), and concludes with the rapid accumulation of mature elastase (33 kDa) in the extracellular environment. In this study, mutants of P. aeruginosa were constructed by gene replacement which expressed lasB1, an allele altered in vitro at an active-site His-223-encoding codon. The lasB1 allele was exchanged for chromosomal lasB sequences in two strain backgrounds, FRD2 and PAO1, through a selectable-cassette strategy which placed a downstream Tn501 marker next to lasB1 and provided the selection for homologous recombination with the chromosome. Two lasB1 mutants, FRD720 and PDO220, were characterized, and their culture supernatants contained greatly reduced proteolytic (9-fold) and elastolytic (14- to 20-fold) activities compared with their respective parental lasB+ strains. This was primarily due to the effect of His-223 substitution on substrate binding by elastase and thus its proteolytic activity. However, the concentration of supernatant elastase antigen was also reduced (five- to sevenfold) in the mutant strains compared with the parental strains. An immunoblot analysis of cell extracts showed a large accumulation of 51-kDa proelastase within lasB1 mutant cells which was not seen in wild-type cell extracts. A time course study showed that production of extracellular elastase was inefficient in the lasB1 mutants compared with that of parental strains. This showed that expression of an enzymatically defective elastase inhibits proper processing of proelastase and provides further evidence for autoproteolytic processing of proelastase in P. aeruginosa. Unlike the parental strains, culture supernatants of the lasB1 mutants contained two prominent elastase species that were 33 and 36 kDa in size. Extracellular 51-kDa proelastase was barely detectable, even though it accumulated to high concentrations within the lasB1 mutant cells. These data suggest that production of an enzymatically defective elastase affects proper secretion because autoproteolytic processing of proelastase is necessary for efficient localization to the extracellular milieu. The appearance of reduced amounts of extracellular elastase and their sizes of 33 and 36 kDa suggest that lasB1-encoded elastase was processed by alternate, less-efficient processing mechanisms. Thus, proelastase must be processed by removal of nearly all of the 18-kDa propeptide before elastase is a protein competent for extracellular secretion.

Alleles↗
PubMed1998

Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440.

A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strain E. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter, bolAp1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of the nlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele of rpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultant rpoS mutant of P.putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein of P. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

Amino Acid Sequence↗
PubMed1999

Induction of AIDS in rhesus monkeys by a recombinant simian immunodeficiency virus expressing nef of human immunodeficiency virus type 1.

A nef gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the nef genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent nef alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1 nef sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient nef allele, and a nef consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1 nef alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open nef reading frame or optimized the HIV-1 nef translational context. The HIV-1 nef gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1 nef can substitute for SIVmac nef in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight.

To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1beta-gal/lbeta-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.

Animals↗
PubMed1986

Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

Cloning, Molecular↗
PubMed1989

Relationships of milk protein types to lifetime performance.

Data from 889 cows at five research stations of Agriculture Canada were used to study the effects of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin loci on herdlife and total yield over fixed parities (one, two, and three parity) and to a fixed age (36, 48, and 61 mo). Actual yields of all cows were utilized to compute total milk regardless of lactational length. The model consisted of station, breed, year of birth, season of birth, and milk protein types with age at first calving as a covariate. Of the four milk protein types studied, only the kappa-casein locus had significant effects on fixed parity and fixed age total milk and herdlife. Cows with BB kappa-casein type outproduced those with AB or AA kappa-casein types in three parity total milk by 963 and 1657 kg, respectively. Considering total milk accumulated up to 61 mo of age in life, cows with BB kappa-casein type outperformed their counterparts with AB or AA kappa-casein types by 1050 and 1923 kg, respectively. Complete replacement of A by B allele at kappa-casein locus would result in an increase of 1657 kg in three parity total milk and an increase of 1923 kg in 61-mo total milk. The moderate gene frequency of kappa-casein B allele in the current dairy population can be increased to improve lifetime total milk to the benefit of the dairy industry.

Alleles↗
PubMed1999

Introduction of Human Chromosome 13 into Retinoblastoma-Negative Metastatic Human Prostate Cancer Cells Increases Their Sensitivity to Growth Inhibition by Transforming Growth Factor-&be;1.

Like many other carcinomas, prostate cancer develops resistance to inhibition by transforming growth factor (TGF)-&be;1 during oncogenesis. One proposed mechanism of TGF-&be;1 action posits action of the retinoblastoma protein (pRb) to suppress c-myc transcription to inhibit cellular proliferation. A metastatic human prostate cancer cell line, DU145, has both nonfunctional pRb and markedly reduced sensitivity to TGF-&be;1 growth inhibition. The defective rb gene in DU145 cells was replaced by a normal rb allele by microcell fusion of chromosome 13. Two subclones, DU145-Cl-I and DU145-Cl-II, were studied in vitro to determine whether the pRb restoration increased sensitivity to the inhibitory effects of TGF-&be;1. By reverse transcriptase-polymerase chain reaction, increased sensitivity to the inhibitory effects of TGF-&BE;1. By reverse transcriptase-polymerase chain reaction, parental DU145 cells had TGF-b receptors of Type I and Type II. Introduction of chromosome 13 reduced the growth rate and prolonged the G1 phase compared with the parental DU145 cell line. Moreover, responsiveness to TGF-&be;1 growth inhibition was restored in a dose-dependent manner. Transcription of c-myc was not altered by TGF-&be;1 growth inhibition. Thus, DU145 cells presumably required the presence of wildtype rb to become growth inhibited that is independent of c-myc transcription. As the entire chromosome 13 was introduced, unknown tumor suppressor genes, not only rb, may be responsible for the restoration of TGF-&be;1 growth inhibition.

Journal Article↗
PubMed1996

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila.

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.

Alleles↗
PubMed2006

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2).

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.

Alleles↗
PubMed1994

Effect of trinucleotide repeat length and parental sex on phenotypic variation in spinocerebellar ataxia I.

Trinucleotide repeat expansion has been found in 64 subjects from 19 families: 57 patients with SCA1 and 7 subjects predicted, by haplotype analysis, to carry the mutation. Comparison with a large set of normal chromosomes shows two distinct distributions, with a much wider variation among expanded chromosomes. The sex of transmitting parent plays a major role in the size distribution of expanded alleles, those with > 54 repeats being transmitted by affected fathers exclusively. Our data suggest that alleles with > 54 repeats have a reduced chance of survival; these appear to be replaced in each generation by further expansion of alleles in the low- to medium-expanded repeat range, preferentially in male transmissions. Detailed clinical follow-up of a subset of our patients demonstrates significant relationships between increasing repeat number on expanded chromosomes and earlier age at onset, faster progression of the disease, and earlier age at death.

Adult↗
PubMed1984

Krüppel, a gene whose activity is required early in the zygotic genome for normal embryonic segmentation.

Embryos homozygous for Krüppel die as late embryos with an altered segmentation pattern. In strong alleles the normal thoracic and anterior abdominal segments are replaced by a partial mirror image duplication of the posterior abdomen. Weak alleles cause smaller pattern deletions in the thorax and abdomen and are not associated with mirror image duplications. The altered segmentation pattern can be traced back to 12 min after the onset of gastrulation, when the shorter germ bands in homozygous Kr embryos provide a first indication of abnormal patterning. The mutant was mapped to position 107.6 at the tip of the right arm of the second chromosome, cytologically to bands 60F2-5. Analysis of homozygous deficiency embryos indicate that the phenotype produced by strong point mutations probably represents the amorphic condition. The requirement for Kr+ gene activity is strictly zygotic. Maternal dosage of Kr+ has no effect on the embryonic phenotype, nor does homozygosity for Kr prevent germ cells from making normal eggs capable of normal embryonic development when fertilized by wild-type sperm. The requirement for Kr+ seems restricted to embryogenesis. Homozygous clones induced in imaginal discs during larval development survive and develop normally and in vivo cultures established from homozygous embryos proliferate normally and metamorphose into adult structures of normal morphology.

Abdomen↗
PubMed1997

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly.

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Alleles↗