Differential distribution of novel restriction-modification systems in clonal lineages of Neisseria meningitidis

Using representational difference analysis, we isolated novel meningococcal restriction-modification (R-M) systems. NmeBI, which is a homologue of the R-M system HgaI of Pasteurella volantium, was present in meningococci of the ET-5 complex and of lineage III. NmeAI was found in serogroup A, ET-37 complex, and cluster A4 meningococci. NmeDI was harbored by meningococci of the ET-37 complex and of cluster A4, but not by serogroup A meningococci. Two of the R-M systems, NmeBI and NmeDI, were located at homologous positions between the phenylalanyl-tRNA synthetase genes pheS and pheT, which appeared to be a preferential target for the insertion of foreign DNA in meningococci. The distribution of the three R-M systems was tested with 103 meningococcal strains comprising 49 sequence types. The vast majority of the strains had either NmeBI, NmeAI, or both NmeAI and NmeDI. Using cocultivation experiments, we could demonstrate that NmeBI, which was present in ET-5 complex meningococci, was responsible for a partial restriction of DNA transfer from meningococci of the ET-37 complex to meningococci of the ET-5 complex.     

Sequence analysis and phenotypes of five temperature sensitive mutator alleles of dnaE, encoding modified alpha-catalytic subunits of Escherichia coli DNA polymerase III holoenzyme.

In the 1970s, several thermosensitive alleles of dnaE (encoding the alpha-catalytic subunit of pol III) were isolated. Genetic characterization of these dnaE mutants revealed that some are mutator alleles at permissive temperature. We have determined the nucleotide changes of five such temperature sensitive mutator alleles (dnaE9, dnaE74, dnaE486, dnaE511, and dnaE1026) and find that most are single missense mutations. The exception is dnaE1026 which is a compound allele consisting of multiple missense mutations. When the previously characterized mutator alleles were moved into a lexA51(Def) recA730 strain, dnaE486, dnaE1026 and dnaE74 conferred a modest approximately two-six-fold increase in spontaneous mutagenesis when grown at the permissive temperature of 28 degrees C, while dnaE9 and dnaE511 actually resulted in a slight decrease in spontaneous mutagenesis. In isogenic DeltaumuDC derivatives, the level of spontaneous mutagenesis dropped significantly, although in each case, the overall mutator effect conferred by the dnaE allele was relatively larger, with all five dnaE alleles conferring an increased spontaneous mutation rate approximately 5-22-fold over the isogenic dnaE+ DeltaumuDC strain. Interestingly, the temperature sensitivity conferred by each allele varied considerably in the lexA51(Def) recA730 background and in many cases, this phenotype was dependent upon the presence of functional pol V (UmuD'2C). Our data suggest that pol V can compete effectively with the impaired alpha-subunit for a 3' primer terminus and as a result, a large proportion of the phenotypic effects observed with strains carrying missense temperature sensitive mutations in dnaE can, in fact, be attributed to the actions of pol V rather than pol III.     

DNA polymerase V-dependent mutator activity in an SOS-induced Escherichia coli strain with a temperature-sensitive DNA polymerase III.

The temperature-sensitive DNA polymerase III (Pol III) encoded by the dnaE486 allele confers a spontaneous mutator activity in SOS-induced bacteria that is largely dependent upon DNA polymerase V (Pol V), encoded by umuD, C. This mutator activity is influenced by the defective proof-reading sub-unit of Pol III encoded by the dnaQ905 (mutD5) allele arguing that Pol V is most likely fixing mutations arising from mismatched primer termini produced by Pol III(486). The size of the dnaQ effect is, however, modest leaving open the possibility that Pol V may be responsible for some of the mutator effect by engaging in bursts of processive activity.     

The establishment of Neisseria meningitidis serogroup W135 of the clonal complex ET-37/ST-11 as an epidemic clone and the persistence of serogroup A isolates in Burkina Faso

We analyzed 48 invasive isolates of Neisseria meningitidis that were isolated from meningitis cases in Burkina Faso (April 2002 to April 2003). Thirty-nine of these isolates had the phenotype (serogroup:serotype:serosubtype) W135:2a:P1.5,2, eight isolates were A:4:P1.9 and one isolate was nongroupable:nonserotypable:nonserosubtypable. Genotyping of meningococcal isolates showed that W135 isolates belonged to the sequence type (ST)-11. The nongroupable isolate was of genogroup W135 and belonged to ST-192. Isolates of serogroup A belonged to ST-2859 (a member of the subgroup III/ST-5 clonal complex). W135 (ST-11) isolates involved in meningitis outbreaks in Burkina Faso differed from those involved in the Hajj-2000 associated outbreak by their pulsed-field gel electrophoresis profile. These data confirm the changing epidemiology of meningococcal infection in Burkina Faso with the establishment and expansion of serogroup W135 N. meningitidis strains of the ET-37/ST-11 clonal complex, as well as the emergence of a new clone within the subgroup III/ST-5 clonal complex.     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

A new mutator strain of Bacillus subtilis

Summary Bacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures. Both chromosomal and plasmid-borne genes were affected by the mutator. Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation. Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).     

Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster

Summary Colony forming ability of Escherichia coli strains carrying the rnh-339::cat mutant allele is strongly dependent on the recBCD and sbcB genes. A mutation inactivating either the RecBCD nuclease or exonuclease I (sbcB) is sufficient to restrict severely the efficiency of plating of strains carrying the rnh-339::cat mutation. Combining a non-lethal temperature-sensitive mutation in the RecBCD nuclease, recB270 (Ts) or recC271 (Ts), with rnh-339::cat renders strains temperature sensitive for growth, even though rnh ⁺ strains with the recB270 (Ts) or recC271 (Ts) alleles are viable at 42 C. The recombinational functions of the RecBCD nuclease can be excluded as the source of lethality on the basis of the following observations. Introduction of a recombination proficient, exonuclease defective recD1009 allele or production of the phage GamS protein (an inhibitor of the RecBCD exonuclease activity) in an rnh-339::cat strain dramatically delays or impairs the ability of such strains to form colonies. Restoration of recombination proficiency by inclusion of an sbcB15 mutation with recB21 recC22 mutations does not restore the ability of the rnh-339::cat mutant strains to plate normally. A recBCD ⁺ strain bearing the rnh-339::cat and sbcB15 mutations forms very few visible colonies after 24 h but forms colonies at normal frequencies after 48 h of incubation. Finally, plating efficiencies of strains are unaffected when the RecBCD recombination pathway is inactivated by introduction of recA56 into an rnh-339::cat strain. These results imply that the defective growth of rnh-339::cat recBCD strains is due to a defect in repair and not recombination mediated by either the RecBCD or the RecF pathway.     

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli

Summary Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ ⁺. dnaQ49 is a recessive allele that confers temperature-sensitive and saltsuppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA^*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA^* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA^* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.     

DNA methylation alters the pattern of spontaneous mutation in Escherichia coli cells (mutD) defective in DNA polymerase III proofreading

We have shown previously that dam mutants of Escherichia coli have a weak mutator phenotype which generates mostly transition mutations in the P22 mnt gene. In contrast, in mutD5 cells, which have a strong mutator phenotype, transversion mutations were the most prevalent. A dam-16 mutD5 strain, defective in both DNA polymerase III associated-proofreading and Dam-directed mismatch repair exhibits a strong mutator phenotype but, surprisingly, its mutation spectrum is similar to that of the dam rather than the mutD parent. The most likely explanation is that Dam-directed mismatch repair in the mutD5 strain corrects most of the potential transition mutations (therefore yielding transversions) in the newly synthesised strand. When the dam-16 allele is present together with mutD5 a reduced efficiency of repair as well as loss of strand discrimination and misdirected repair results in the appearance of transition mutations at high frequency.     

Kuraishia MolischianaSp. Nov., the Teleomorph of Candida Molischiana

Thirty-two strains, many of them isolated from wood-associated habitats, and designated as Kuraishia (Pichia) capsulata and Candida molischiana according to their phenotype, exhibited two types of HaeIII restriction fragment patterns of their small subunit rDNA with the neighboring ITS. One fragment pattern corresponded to that of the type strain of K. capsulata, whereas the other pattern was unique to the typestrain of C. molischiana. Sequencing of the D1/D2 domain of the large subunit rDNA confirmed that the different HaeIII restriction fragment patterns of small subunit rDNA with the neighboring ITS reliably distinguished K. capsulata from C. molischiana. Ascospore formation was observed in several C. molischiana strains and K. molischiana (type strain: NCAIM Y.01725, CBS 9993) is proposed as the teleomorphic state of Candida molischiana.     

Oenothera chloroplast DNA polymorphisms associated with plastome mutator activity

Summary Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Chloroplast DNA (cpDNA) was isolated from nine mutants and two green isolates of the plastome mutator line. cpDNA restriction patterns were compared to cpDNA from a representative of the progenitor Johansen strain, and cpDNAs from all eleven plastome mutator lines show changes of fragment mobility due to deletion events at five discrete regions of the plastome. Most of the mutants have cpDNA restriction patterns identical to that of one of the green isolates from the plastome mutator line, and therefore, most of the differences in fragment length are probably not responsible for the mutant phenotypes. In contrast to the plastome mutator line, cpDNA from several populations of a closely related wild-type Oenothera species have few restriction fragment length polymorphisms. This suggests that both mutation frequencies and site-specific cpDNA deletions are elevated in the plastome mutator line, and implicates a defect in the cpDNA repair or replication machinery.     

Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations

Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer. In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function. First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation. Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function. Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis. Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations. Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented. Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain. This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.     

Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype

Expression of serogroup B meningococcal capsular polysaccharide undergoes frequent phase variation involving reversible frameshift mutations within a homopolymeric repeat in the siaD gene. A high rate of phase variation is the consequence of a biochemical defect in methyl-directed mismatch repair. The mutator phenotype is associated to the absence of DNA adenine methyltransferase (Dam) activity in all pathogenic isolates and in 50% of commensal strains. Analysis of the meningococcal dam gene region revealed that in all Dam- strains a gene encoding a putative restriction endonuclease (drg) that cleaves only the methylated DNA sequence 5'-GmeATC-3' replaced the dam gene. Insertional inactivation of the dam and/or drg genes indicated that high rates of phase variation and hypermutator phenotype are caused by absence of a functional dam gene.     

The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a DeltarecA strain raise the possibility that asp --> gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp --> gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC :lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp --> gly misinsertion occurs in recA- cells and, therefore, the requirement for recA is 'downstream' of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.     

Description of Gibbsiella quercinecans gen. nov., sp. nov., associated with Acute Oak Decline

Gram-negative, facultatively anaerobic bacterial strains were consistently isolated from oak trees displaying symptoms of extensive stem bleeding. In Britain, this disorder is called Acute Oak Decline (AOD). A similar condition has been noted on species of Mediterranean oak in Spain. The identity of bacterial isolates from symptomatic trees in both countries was investigated using molecular techniques and phenotypic assays. 16S rRNA gene sequencing indicated that the strains were most closely related to the genera Serratia, Kluyvera, Klebsiella and Raoultella (all>97%). Phylogenetic analysis revealed that the strains formed a distinct lineage within the family Enterobacteriaceae, which was confirmed by both gyrB- and rpoB-gene sequencing. DNA-DNA hybridization confirmed that the strains belonged to a single taxon which could also be differentiated phenotypically from its closest phylogenetic neighbours. The phylogenetic and phenotypic data both demonstrated that the strains isolated from oak represented a novel genus and species within the family Enterobacteriaceae for which the name Gibbsiella quercinecans gen. nov., sp. nov. (type strain=FRB 97(T)=LMG 25500(T)=NCPPB 4470(T)) is proposed.     

Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair

Summary A new recombination gene called recR has been identified and located near dnaZ at minute 11 on the current linkage map of Escherichia coli. The gene was detected after transposon mutagenesis of a recB sbcB sbcC strain and screening for insertion mutants that had a reduced efficiency of recombination in Hfr crosses. The recR insertions obtained conferred a recombination deficient and extremely UV sensitive phenotype in both recB recC sbcA and recB recC sbcB sbcC genetic backgrounds. recR derivatives of recBC ⁺ sbc ⁺ strains were proficient in conjugational and transductional recombination but deficient in plasmid recombination and sensitive to UV light. Strains carrying recR insertions combined with mutations uvrA and other rec genes revealed that the gene is involved in a recombinational process of DNA repair that relies also on recF and recO, and possibly recJ, but which is independent of recB, recC and recD. The properties of two other insertions, one located near pyrE and the other near guaA, are discussed in relation to their proximity to recG and xse (the gene for exonuclease VII), respectively.     

Role of hypermutability on bacterial fitness and emergence of resistance in experimental osteomyelitis due to Staphylococcus aureus

This study was designed to investigate the role of hypermutability of Staphylococcus aureus on bacterial fitness and antibiotic resistance in a model of chronic bone infection. An isogenic pair of strains, S. aureus RN4220 and its mutator counterpart inactivated in the mutL gene were used in a rat model of osteomyelitis of the tibia. The effect of the mutator phenotype in the emergence of antibiotic resistance was assessed in rats infected by each strain separately and treated with rifampicin for 5 days. No difference between the two strains was found in bacterial growth in vitro and in bacterial survival in the animal model, indicating no fitness defect in the mutator strain. In competition studies performed in rats coinfected with the two strains at a same ratio and sacrificed at different times from day 3 to day 42 postinoculation, the mutator strain was clearly disadvantaged because it was found in all rats and at all study times at lower counts (P<0.05 from day 14 to day 42). Two of the 16 rats infected by the mutator strain and none of the 14 rats infected by the wild-type strain had acquired rifampicin-resistant mutants (P=0.4). Data suggest that the S. aureus mutator phenotype was associated with a decreased bacterial fitness in vivo and did not confer significant advantage in the acquisition of antibiotic resistance in a model of chronic bone infection.     

Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex

Capsule switching in Neisseria meningitidis is thought to occur by horizontal DNA exchange between meningococcal strains. Antigenic variants may be generated by allelic replacement of the siaD gene; the variants may then be selected by specific immunity against the capsular antigen. There were several vaccination campaigns against serogroup C in France in 2002, following an increase in the prevalence of invasive isolates of serogroup C of the phenotype C:2a:P1.5 and C:2a:P1.5,2 belonging to the ET-37/ST-11 clonal complex. We evaluated the emergence of capsule variants by the detection of B:2a:P1.5 and B:2a:P1.5,2 meningococcal isolates of the ET-37/ST-11 clonal complex. These isolates were significantly more frequent after the year 2002. Pulsed field gel electrophoresis profiles of the serogroup B (ET-37/ST-11) isolates differed from that of serogroup C (ET-37/ST-11) isolates by the bands that harbor the siaD genes responsible for the serogroup specificity. However, serogroup B and C, ET37/ST-11 isolates both express similar virulence as assessed from colonization and invasiveness in a mouse model. Our results indicate that capsule switching events within the same clonal complex can arise frequently with no alteration in virulence. This justifies an enhanced system of surveillance by molecular typing of such isolates, particularly after serogroup-specific vaccination.     

Genetic diversity of Neisseria meningitidis strains isolated in Rio de Janeiro, Brazil, evaluated by multilocus enzyme electrophoresis

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.     

Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation

Cupriavidus metallidurans CH34 is a β-proteobacterium well equipped to cope with harsh environmental conditions such as heavy metal pollution. The strain carries two megaplasmids specialized in the response to heavy metals and a considerable number of genomic islands, transposons and insertion sequence (IS) elements. The latter were characterized in detail in this study, which revealed nine new IS elements totaling to 21 distinct IS elements from 10 different IS families and reaching a total of 57 intact IS copies in CH34. Analysis of all fully sequenced bacterial genomes revealed that relatives of these IS elements were mostly found in the Burkholderiaceae family (β-proteobacteria) to which C. metallidurans belongs. Three IS elements were 100% conserved in other bacteria suggesting recent interaction and horizontal transfer between these strains. In addition, a number of these IS elements were associated with genomic islands, gene inactivation or rearrangements that alter the autotrophic growth capacities of CH34. The latter rearrangements gave the first molecular evidence for the mutator phenotype that is characteristic for various C. metallidurans strains. Furthermore, differential expression of some IS elements (or adjacent genes in the same strand orientation) was found under heavy metal stress, an environmental stress to which C. metallidurans CH34 is well adapted. These observations indicate that these IS elements play an active role in C. metallidurans CH34 lifestyle, including its metabolic potential and adaptation under selective pressure.