Molecular cloning of two linked loci that increase the transformability of transformation-deficient mutants of Haemophilus influenzae.

A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components.     

Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny.

Mutants of Pseudomonas aeruginosa with impaired ability to establish a lysogenic relationship with temperate bacteriophage (Les-) have been isolated. These les mutations map to two areas of the P. aeruginosa chromosomal map as determined by conjugational and transductional analyses. Two phenotypic classes of Les- mutants were identified. One class of mutations has pleiotropic effects on DNA metabolism. These mutants are unable to recombine genetic material acquired as a result of either conjugation or transduction (Rec-). In addition, the ability of these Les- Rec- mutants to repair UV-induced damage to bacteriophage is reduced (host-cell reactivation deficient, Hcr-). Mutants of the second class are Les-, Rec+, and Hcr+.     

Molecular insights on DNA delivery into Saccharomyces cerevisiae

Understanding of the molecular system for DNA delivery into eucaryotic cells, a key to human DNA therapy, remains obscure. To understand this system, we undertook a study using the Saccharomyces cerevisiae model into which DNA delivery is easily assessed through competence (transformability) and for which all nonessential gene mutants (about 5000 strains) are available. We analyzed the competence of each of these mutants and identified three low-competence mutants, i.e., sin3Delta, she4Delta, and arc18Delta, and three high-competence mutants, i.e., pde2Delta, spf1Delta, and pmr1Delta. Through further studies using the six mutants, we concluded that the Arp2/3 activation machinery involving the Myo3/5p, Vrp1p, Las17p, Pan1p, and Arp2/3 complex is crucial to delivery (competence), and that high cAMP enhances competence via protein kinase A installing Tpk3p. We also propose that DNA is taken up via an endocytosis-like event, being at least partially different from well-known endocytosis.     

Properties of Haemophilus influenzae mutants that are slightly recombination deficient and carry a mutation in the rec-1 gene region.

The highly recombination-deficient rec-1 mutants of Haemophilus influenzae are, as far as tested, equivalent to recA mutants of Escherichia coli. By selection for mutations in the rec-1 gene of H. influenzae, mutants designated ird (intermediary recombination-deficient) mutants were isolated; these mutants were much less recombination deficient (degree of transformability, 0.2 to 30% of wild-type value) than previously isolated rec-1 mutants (degree of transformability, 0.0001% of wild-type value). The ird mutants were more sensitive to ultraviolet irradiation and mytomycin C treatment than the wild type, but less sensitive than rec-1 mutants. Spontaneous production of phage HP1c1 by lysogenic MC11 cells and prophage induction by mitomycin C or ultraviolet irradiation were the same as in the wild type. In the ird mutants endogenous deoxyribonucleic acid was degraded both spontaneously and after ultraviolet irradiation to the same extent as in the wild type. Examination of one of the ird mutants revealed that recombination could be enhanced by ultraviolet irradiation, possibly because of an increased synthesis of the rec-1 gene product induced by ultraviolet irradiation.     

Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.     

Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS‐DegU two‐component system. Here we report a role for flagella in the regulation of the K‐state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU‐P, which inhibits the expression of ComK, the master regulator for the K‐state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hag^A233V) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU‐P levels through an unknown signaling mechanism. This flagellar‐load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K‐state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.     

Transformability of Streptomycin-resistant Group H Streptococci

Several resistant mutants of a transformable group H streptococcus, strain Challis, were isolated from media containing high concentrations of streptomycin. Mutants SR5a and SR5 exhibited high and low transformability, respectively, when exposed to deoxyribonucleic acid (DNA) from a novobiocin-resistant Challis strain. With similar exposure, mutant SR30 exhibited loss of transformability. The mutants further differed from the parent strain in time of appearance of optimal competence, and, in the case of SR5 and SR30, total growth was somewhat less than that of the parent. The rapidity with which transformants appeared upon initial exposure to DNA was approximately the same in the mutants and the parent strain. The decrease or loss of transformability of mutants SR5 and SR30 was found to be due to an alteration in capacity to take up DNA. Mutant SR5a (highly transformable) was further differentiated from mutants SR5 and SR30 in that it was somewhat more sensitive to high concentrations of streptomycin. Transformants obtained by treating strain Challis with the three types of mutant DNA, on the other hand, exhibited similar degrees of resistance to increasing concentrations of streptomycin. The additional decrease in transforming ability of mutant SR5a and the loss of transforming ability of mutant SR5 after a second exposure to streptomycin may indicate a stepwise process in the change from transformability to nontransformability. Although streptomycin resistance may not be directly related to inability to transform, results indicate that streptomycin greatly increases the chances of selecting these mutants and also can be of value in serving as a marker in studies of this nature.     

Location of functional regions of the Escherichia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants.

In previous work (E. S. Tessman and P. K. Peterson, J. Bacteriol. 163:677-687 and 688-695, 1985), we isolated many novel protease-constitutive (Prtc) recA mutants, i.e., mutants in which the RecA protein was always in the protease state without the usual need for DNA damage to activate it. Most Prtc mutants were recombinase positive and were designated Prtc Rec+; only a few Prtc mutants were recombinase negative, and those were designated Prtc Rec-. We report changes in DNA sequence of the recA gene for several of these mutants. The mutational changes clustered at three regions on the linear RecA polypeptide. Region 1 includes amino acid residues 25 through 39, region 2 includes amino acid residues 157 through 184, and region 3 includes amino acid residues 298 through 301. The in vivo response of these Prtc mutants to different effectors suggests that the RecA effector-binding sites have been altered. In particular we propose that the mutations may define single-stranded DNA- and nucleoside triphosphate-binding domains of RecA, that polypeptide regions 1 and 3 comprise part of the single-stranded DNA-binding domain, and that polypeptide regions 2 and 3 comprise part of the nucleoside triphosphate-binding domain. The overlapping of single-stranded DNA- and nucleoside triphosphate-binding domains in region 3 can explain previously known complex allosteric effects. Each of four Prtc Rec- mutants sequenced was found to contain a single amino acid change, showing that the change of just one amino acid can affect both the protease and recombinase activities and indicating that the functional domains for these two activities of RecA overlap. A recA promoter-down mutation was isolated by its ability to suppress the RecA protease activity of one of our strong Prtc mutants.     

Reversible changes of competence of streptococci of the H group

Erythromycin-resistant and oleandomycin-resistant mutants ofStreptococcus challis 560 were obtained. As compared with the parent strains the mutants exhibited a lower transformability. This change of competence is reversible and may be reversed by adding the competence factor released by the parent strain into the supernatant fluid.     

Competence proteins in Bacillus subtilis com mutants

The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these com mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.     

PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae

Neisseria gonorrhoeae, the Gram-negative aetiological agent of gonorrhoea, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp-dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal pilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tfp. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in pilT. Piliated pilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence-specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT-deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.     

Construction of an Agrobacterium tumefaciens C58 recA mutant.

Clones encoding the recA gene of Agrobacterium tumefaciens C58 were isolated from a cosmid bank by complementation of an Escherichia coli recA mutation. Subcloning and mutagenesis with the lacZ fusion transposon Tn3HoHo1 located the Agrobacterium recA gene to a 1.3-kilobase segment of DNA. beta-Galactosidase expression from the fusions established the direction in which the gene was transcribed. The gene restored homologous recombination as well as DNA repair functions in E. coli recA mutants. Similar complementation of DNA repair functions was observed in the UV-induced Rec- Agrobacterium mutant, LBA4301. The Agrobacterium recA gene was disrupted by insertion of a cassette encoding resistance to erythromycin, and the mutated gene was marker exchanged into the chromosome of strain NT-1. The resulting strain, called UIA143, was sensitive to UV irradiation and methanesulfonic acid methyl ester and unable to carry out homologous recombination functions. The mutation was stable and had no effect on other genetic properties of the Agrobacterium strain, including transformability and proficiency as a conjugal donor or recipient. Furthermore, strain UIA143 became tumorigenic upon introduction of a Ti plasmid, indicating that tumor induction is independent of recA functions. Sequence homology was detected between the recA genes of strain C58 and E. coli as well as with DNA isolated from agrobacteria representing the three major biochemically differentiated biovars of this genus. In some cases, biovar-specific restriction fragment length polymorphisms were apparent at the recA locus.     

Relationships between a Hyper-Rec Mutation (rem1 ) and Other Recombination and Repair Genes in Yeast

Mutations in the REM1 gene of Saccharomyces cerevisiae confer a semidominant hyper-recombination and hypermutable phenotype upon mitotic cells ( GOLIN and ESPOSITO 1977). These effects have not been observed in meiosis. We have examined the interactions of rem1 mutations with rad6-1, rad50 -1, rad52-1 or spo11 -1 mutations in order to understand the basis of the rem1 hyper-rec phenotype. The rad mutations have pleiotropic phenotypes; spo11 is only defective in sporulation and meiosis. The RAD6, RAD50 and SPO11 genes are not required for spontaneous mitotic recombination; mutations in the RAD52 gene cause a general spontaneous mitotic Rec- phenotype. Mutations in RAD50 , RAD52 or SPO11 eliminate meiotic recombination, and mutations in RAD6 prevent spore formation. Evidence for the involvement of RAD6 in meiotic recombination is less clear. Mutations in all three RAD genes confer sensitivity to X rays; the RAD6 gene is also required for UV damage repair. To test whether any of these functions might be involved in the hyper-rec phenotype conferred by rem1 mutations, double mutants were constructed. Double mutants of rem1 spo11 were viable and demonstrated rem1 levels of mitotic recombination, suggesting that the normal meiotic recombination system is not involved in producing the rem1 phenotype. The rem1 rad6 double mutant was also viable and had rem1 levels of mitotic recombination. Neither rem1 rad50 nor rem1 rad52 double mutants were viable. This suggests that rem1 causes its hyper-rec phenotype because it creates lesions in the DNA that are repaired using a recombination-repair system involving RAD50 and RAD52.     

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.     

Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12

We isolated recA mutants with altered protease activity and then examined recombinase activity to determine whether the protease and recombinase functions of the RecA protein of Escherichia coli are separable. We found five mutants that had moderately strong constitutive RecA protease activity but no recombinase activity above the delta recA strain background, the first clear-cut examples of mutants of this class, designated Prtc Rec-. We also isolated 65 mutants that were protease-defective toward the LexA repressor and found that all of them were also recombinase deficient. Four of these mutants retained both partial recombinase activity and partial inducible protease activity. The recombinase-defective mutants were much more sensitive than the recA+ strain to crystal violet, kanamycin, and chloramphenicol, indicating altered membrane permeability. The recA (Prtc Rec-) mutants had a subtle alteration in protease specificity, all being defective in spontaneous induction of phages lambda imm434 and 21. They differed from Prtc Rec+ mutants of comparable or even weaker constitutive protease strength, all of which showed dramatic spontaneous induction of these prophages. However, treating a Prtc Rec- mutant with mitomycin C resulted in significant prophage induction. Thus, the RecA proteins of the Prtc Rec- mutants have constitutive protease activity toward the LexA repressor, but have only DNA damage-activable protease activity toward phage repressors. UV-induced mutagenesis from his to his+ was studied for one Prtc Rec- mutant, and induced mutation frequencies as high as those for the recA+ strain were found despite the absence of recombinase activity.