Alteration of flagellar phenotype of Escherichia coli strain P12b, the standard type strain for flagellar antigen H17, possessing a new non-fliC flagellin gene flnA, and possible loss of original flagellar phenotype and genotype in the course of subculturing through semisolid media

A practically important phenomenon, resulting in the loss of the original flagellar phenotype (genotype) of bacteria, is described in the Escherichia coli H17 type strain P12b possessing two distinct genes for H17 and H4 flagellins, respectively. By PCR, sequencing, and phylogenetic investigation, the H17 gene (originally expressed) was considered a new non-fliC flagellin gene and assigned flnA, while the H4 gene (originally cryptic) was reaffirmed as fliC. H17 and H4 flagella differed morphologically. The phenomenon consisted in the replacement of H17 cells by H4 cells during subculturing through certain semisolid media and resulted from the excision of flnA _H17 entirely or in part. The substitution rate depended on the density and nutrient composition of media and reached 100% even after a single passage through 0.3% LB agar. Such phenomenon can lead to an unexpected loss of original H17 phenotype. Our review of the literature showed that the loss of the original flagellar genotype (phenotype) of P12b has occurred in some laboratories while the authors continued to consider their cultures H17. We showed how to distinguish these alternative flagellin genotypes using popular fliC primers. Attention was also paid to possible discrepancies between serological and molecular results in flagellar typing of E. coli.     

Site-specific recombination Xis-independent excisive recombination of bacteriophage lambda

The integration of bacteriophage lambda into the Escherichia coli chromosome depends on the phage-encoded Int protein; prophage excision depends on Int and a second phage function, Xis. Limited excisive recombination has been observed in vivo with certain xis mutants, suggesting that Int may be able to carry out excision without Xis.We report here that purified Int protein carries out lambda site-specific excisive recombination in vitro in the absence of Xis. This reaction requires host factors derived from a non-lysogenic E. coli strain and is influenced strongly by ionic strength. Excision in the absence of Xis occurs slowly at low salt concentrations (40 mm-NaCl) and very little excision occurs at high salt concentrations (100 mm-NaCl). In the presence of Xis, excisive recombination proceeds rapidly at both low and high ionic strengths. These observations are consistent with previous experiments that suggested the partial dispensability of Xis for excision.     

Control of Staphylococcus aureus pathogenicity island excision

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15–17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small‐headed phage‐like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI‐encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI‐coded Xis is absolutely required for recombination between the SaPI att_L and att_R sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.     

Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai

Prophages make up 12% of the enterohemorrhagic Escherichia coli genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of E. coli O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 × 10⁻⁴. One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing pchB and psrC, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.     

Microhomology-mediated deletion and gene conversion in African trypanosomes

Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.     

A 32 kb critical region excluding Y402H in CFH mediates risk for age-related macular degeneration

Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10⁻¹⁰⁹); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10⁻⁹) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.     

Pathogenic and commensal Escherichia coli from irrigation water show potential in transmission of extended spectrum and AmpC β‐lactamases determinants to isolates from lettuce

There are few studies on the presence of extended-spectrum β-lactamases and AmpC β-lactamases (ESBL/AmpC) in bacteria that contaminate vegetables. The role of the production environment in ESBL/AmpC gene transmission is poorly understood. The occurrence of ESBL/AmpC in Escherichia coli (n = 46) from lettuce and irrigation water and the role of irrigation water in the transmission of resistant E. coli were studied. The presence of ESBL/AmpC, genetic similarity and phylogeny were typed using genotypic and phenotypic techniques. The frequency of β-lactamase gene transfer was studied in vitro. ESBLs/AmpC were detected in 35 isolates (76%). Fourteen isolates (30%) produced both ESBLs/AmpC. Prevalence was highest in E. coli from lettuce (90%). Twenty-two isolates (48%) were multi-resistant with between two and five ESBL/AmpC genes. The major ESBL determinant was the CTX-M type (34 isolates). DHA (33% of isolates) were the dominant AmpC β lactamases. There was a high conjugation efficiency among the isolates, ranging from 3.5 × 10⁻² to 1 × 10⁻² ± 1.4 × 10⁻¹ transconjugants per recipient. Water isolates showed a significantly higher conjugation frequency than those from lettuce. A high degree of genetic relatedness between E. coli from irrigation water and lettuce indicated possible common ancestry and pathway of transmission.     

New Flagellin-Specifying Genes in Some Escherichia coli Strains

Data for further development of the flagellar antigen genetics of the species Escherichia coli are reported. Two new flagellin genes named fllA and flmA were found in E. coli 781-55, E2987-73, and E223-69, the test strains for E. coli flagellar antigens H44, H55, and H54, respectively (collection of the International Escherichia and Klebsiella Centre of the World Health Organization, Copenhagen, Denmark). Two alleles of fllA were identified that encode flagellar antigens H44 (fllA₄₄) and H55 (fllA₅₅), and the only flmA allele found (flmA₅₄) encodes antigen H54. The sites of their integration in the E. coli K-12 chromosome after P1-mediated transduction were approximately determined and found to be separate from each other and from the known regions of flagellar genes of E. coli and salmonellae. The region of flm₅₄ was found to repress the expression of some alleles of the flagellin gene fliC. In addition, cryptic genes encoding antigens H4 and H38 were found in phenotypically monophasic test strains 781-55 and E2987-73, respectively.     

A genomic islet mediates flagellar phase variation in Escherichia coli strains carrying the flagellin-specifying locus flk

The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the flk region between the chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the flk flagellin-specifying gene flkA and a fliC repressor gene, flkB, indicating that there is a typical genomic islet (GI), which was designated the flk GI. The horizontal transfer potential of the flk GI was indicated by detection of the excised extrachromosomal circular form of the flk GI. By generating fliC-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be expressed as fliC(off) + flkA(on) --> fliC(on) + flkA(none). This is the first time that a molecular mechanism for flagellar phase variation has been reported for E. coli.     

Recombination between repeats in Escherichia coli by a recA-independent,proximity-sensitive mechanism

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 × 10^?5 per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.     

Non-recurrent 17p11.2 deletions are generated by homologous and non-homologous mechanisms

Several recurrent common chromosomal deletion and duplication breakpoints have been localized to large, highly homologous, low-copy repeats (LCRs). The mechanism responsible for these rearrangements, viz., non-allelic homologous recombination between LCR copies, has been well established. However, fewer studies have examined the mechanisms responsible for non-recurrent rearrangements with non-homologous breakpoint regions. Here, we have analyzed four uncommon deletions of 17p11.2, involving the Smith–Magenis syndrome region. Using somatic cell hybrid lines created from patient lymphoblasts, we have utilized a strategy based on the polymerase chain reaction to refine the deletion breakpoints and to obtain sequence data at the deletion junction. Our analyses have revealed that two of the four deletions are a product of Alu/Alu recombination, whereas the remaining two deletions result from a non-homologous end-joining mechanism. Of the breakpoints studied, three of eight are located in LCRs, and five of eight are within repetitive elements, including Alu and MER5B sequences. These findings suggest that higher-order genomic architecture, such as LCRs, and smaller repetitive sequences, such as Alu elements, can mediate chromosomal deletions via homologous and non-homologous mechanisms. These data further implicate homologous recombination as the predominant mechanism of deletion formation in this genomic interval.     

Recurrent distal 7q11.23 deletion including HIP1 and YWHAG identified in patients with intellectual disabilities, epilepsy, and neurobehavioral problems

We report 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of a heterozygous microdeletion distally adjacent to the Williams-Beuren syndrome region on chromosome 7q11.23. In six families with a common recurrent ∼1.2 Mb deletion that includes the Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) genes and that is flanked by large complex low-copy repeats, we identified sites for nonallelic homologous recombination in two patients. There were no cases of this ∼1.2 Mb distal 7q11.23 deletion copy number variant identified in over 20,000 control samples surveyed. Three individuals with smaller, nonrecurrent deletions (∼180–500 kb) that include HIP1 but not YWHAG suggest that deletion of HIP1 is sufficient to cause neurological disease. Mice with targeted mutation in the Hip1 gene (Hip1^−/−) develop a neurological phenotype characterized by failure to thrive, tremor, and gait ataxia. Overall, our data characterize a neurodevelopmental and epilepsy syndrome that is likely caused by recurrent and nonrecurrent deletions, including HIP1. These data do not exclude the possibility that YWHAG loss of function is also sufficient to cause neurological phenotypes. Based on the current knowledge of Hip1 protein function and its proposed role in AMPA and NMDA ionotropic glutamate receptor trafficking, we believe that HIP1 haploinsufficiency in humans will be amenable to rational drug design for improved seizure control and cognitive and behavioral function.     

Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5′UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5′ sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.     

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobacillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP?×?attB recombination event. In a cell-free extract of E. coli at 42°?C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trans between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro.     

Adaptive evolution of UGT2B17 copy-number variation

The human UGT2B17 gene varies in copy number from zero to two per individual and also differs in mean number between populations from Africa, Europe, and East Asia. We show that such a high degree of geographical variation is unusual and investigate its evolutionary history. This required first reinterpreting the reference sequence in this region of the genome, which is misassembled from the two different alleles separated by an artifactual gap. A corrected assembly identifies the polymorphism as a 117 kb deletion arising by nonallelic homologous recombination between ~4.9 kb segmental duplications and allows the deletion breakpoint to be identified. We resequenced ~12 kb of DNA spanning the breakpoint in 91 humans from three HapMap and one extended HapMap populations and one chimpanzee. Diversity was unusually high and the time to the most recent common ancestor was estimated at ~2.4 or ~3.0 million years by two different methods, with evidence of balancing selection in Europe. In contrast, diversity was low in East Asia where a single haplotype predominated, suggesting positive selection for the deletion in this part of the world.     

Replicative resolution of integron cassette insertion

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC × attI reaction.     

Population-specific recombination sites within the human MHC region

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.     

A defined terminal region of the E. coli chromosome shows late segregation and high FtsK activity

Background

The FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV.

Methodology

We have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a ∼400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPS-recognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions.

Significance

Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last steps of chromosome segregation and to their coupling with cell division by FtsK.     

How Hot Are Drosophila Hotspots? Examining Recombination Rate Variation and Associations with Nucleotide Diversity,Divergence, and Maternal Age in Drosophila pseudoobscura

Fine scale meiotic recombination maps have uncovered a large amount of variation in crossover rate across the genomes of many species, and such variation in mammalian and yeast genomes is concentrated to <5kb regions of highly elevated recombination rates (10–100x the background rate) called “hotspots.” Drosophila exhibit substantial recombination rate heterogeneity across their genome, but evidence for these highly-localized hotspots is lacking. We assayed recombination across a 40Kb region of Drosophila pseudoobscura chromosome 2, with one 20kb interval assayed every 5Kb and the adjacent 20kb interval bisected into 10kb pieces. We found that recombination events across the 40kb stretch were relatively evenly distributed across each of the 5kb and 10kb intervals, rather than concentrated in a single 5kb region. This, in combination with other recent work, indicates that the recombination landscape of Drosophila may differ from the punctate recombination pattern observed in many mammals and yeast. Additionally, we found no correlation of average pairwise nucleotide diversity and divergence with recombination rate across the 20kb intervals, nor any effect of maternal age in weeks on recombination rate in our sample.     

RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived ‘MgPar’ donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10^?4 events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption‐deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C–MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.