Functional significance of allelic variation at methuselah, an aging gene in Drosophila

Background

Longevity and age-specific patterns of mortality are complex traits that vary within and among taxa. Multiple candidate genes for aging have been identified in model systems by extended longevity mutant phenotypes, including the G-protein coupled receptor methuselah (mth) in D. melanogaster. These genes offer important insights into the mechanisms of lifespan determination and have been major targets of interest in the biology of aging. However, it is largely unknown whether these genes contribute to genetic variance for lifespan in natural populations, and consequently contribute to lifespan evolution.

Methodology/Principle Findings

For a gene to contribute to genetic variance for a particular trait, it must meet two criteria: natural allelic variation and functional differences among variants. Previous work showed that mth varies significantly among wild populations; here we assess the functional significance of wild-derived mth alleles on lifespan, fecundity and stress resistance using a quantitative complementation scheme. Our results demonstrate that mth alleles segregating in nature have a functional effect on all three traits.

Conclusions/Significance

These results suggest that allelic variation at mth contributes to observed differences in lifespan and correlated phenotypes in natural populations, and that evaluation of genetic diversity at candidate genes for aging can be a fruitful approach to identifying loci contributing to lifespan evolution.     

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.     

Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val¹⁰⁹Ile, Asp³³⁵Glu and Asn³⁸⁴Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.     

Genetic structure and contrasting selection pattern at two major histocompatibility complex genes in wild house mouse populations

The mammalian major histocompatibility complex (MHC) is a tightly linked cluster of immune genes, and is often thought of as inherited as a unit. This has led to the hope that studying a single MHC gene will reveal patterns of evolution representative of the MHC as a whole. In this study we analyse a 1000-km transect of MHC variation traversing the European house mouse hybrid zone to compare signals of selection and patterns of diversification at two closely linked MHC class II genes, H-2Aa and H-2Eb. We show that although they are 0.01 cM apart (that is, recombination is expected only once in 10 000 meioses), disparate evolutionary patterns were detected. H-2Aa shows higher allelic polymorphism, faster allelic turnover due to higher mutation rates, stronger positive selection at antigen-binding sites and higher population structuring than H-2Eb. H-2Eb alleles are maintained in the gene pool for longer, including over separation of the subspecies, some H-2Eb alleles are positively and others negatively selected and some of the alleles are not expressed. We conclude that studies on MHC genes in wild-living vertebrates can give substantially different results depending on the MHC gene examined and that the level of polymorphism in a related species is a poor criterion for gene choice.     

Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of th proA step.

Two chromosomal loci containing the Corynebacterium glutamicum ATCC 17965 proB and proC genes were isolated by complementation of Escherichia coli proB and proC auxotrophic mutants. Together with a proA gene described earlier, these new genes describe the major C. glutamicum proline biosynthetic pathway. The proB and proA genes, closely linked in most bacteria, are in C. glutamicum separated by a 304-amino-acid open reading frame (unk) whose predicted sequence resembles that of the 2-hydroxy acid dehydrogenases. C. glutamicum mutants that carry null alleles of proB, proA, and proC were constructed or isolated from mutagenized cultures. Single proC mutants are auxotrophic for proline and secrete delta1-pyrroline-5-carboxylate, which are the expected phenotypes of bacterial proC mutants. However, the phenotypes or proB and proA mutants are unexpected. A proB mutant has a pleiotropic phenotype, being both proline auxotrophic and affected in cell morphology. Null proA alleles still grow slowly under proline starvation, which suggests that a proA-independent bypass of this metabolic step exists in C. glutamicum. Since proA mutants are complemented by a plasmid that contains the wild-type asd gene of C. glutamicum, the asd gene may play a role in this bypass.     

An ‘instant gene bank’ method for gene cloning by mutant complementation

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication (‘helper plasmid’). Transformant colonies appear as the result of the Joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this “instant gene bank” technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.     

sacB-5-Fluoroorotic Acid-pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB—5-fluoroorotic acid (5FOA)—pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura⁻) and hence resistant to 5FOA (5FOA^r). Although Ura⁺ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA^r to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA^r selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA^r colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211–213]. A family of related plasmids containing different antibiotic-resistance markers (Cm^R or Gm^R or Km^R and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.     

Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3′ end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, β-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.     

Phosphoglucose isomerase polymorphism in the shrimp Metapenaeus affinis

Three phenotypes of phosphoglucose isomerase (PGI) were detected in abdominal muscle extracts from the shrimp (Metapenaeus affinis) by starch-gel electrophoresis. The observed phenotypes were assumed to be under the control of two allelic genes. This assumption was supported by the observed distribution of phenotypes. There were no significant differences in the distribution of PGI phenotypes among samples of shrimp taken from Basrah waters, Iraq and from Kuwaiti waters, Arab Gulf. The three PGI alleles observed in M. affinis appear to be the same in the two localities.     

Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.     

Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod–res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.     

Distribution of Allelic Variants of the Chromosomal Gene bla OXA-114-like in Achromobacter xylosoxidans Clinical Isolates

Achromobacter xylosoxidans is increasingly being documented in cystic fibrosis patients. The bla _OXA-114 gene has been recognized as a naturally occurring chromosomal gene, exhibiting different allelic variants. In the population under study, the bla _OXA-114-like gene was found in 19/19 non-epidemiological-related clinical isolates of A. xylosoxidans with ten different alleles including 1 novel OXA-114 variant.     

Development of an integrative DNA transformation system for the yeast Hansenula anomala

A host-vector system for the yeast Hansenula anomala was developed. The system was based on an auxotrophic mutant host of H. anomala which was defective in orotidine-5′-phosphate decarboxylase (ODCase) activity. The H. anomala ODCase-negative mutant strains (ura3 strains) were isolated based on 5-fluoroorotic acid (5-FOA) resistance. A plasmid vector containing the H. anomala URA3 gene was used for transformation. Using this plasmid, all of the H. anomala ura3 strains tested could be transformed to Ura⁺ phenotypes. In all of Ura⁺ transformants, the introduced plasmid was integrated into the chromosomal URA3 locus by homologous recombination. The Ura⁺ phenotype of the transformants was stably maintained after nonselective growth.     

Development and Application of a upp-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM

In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism''s survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp-encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the Δupp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus. The resulting ΔslpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus.Lactobacillus acidophilus NCFM is a commercially established probiotic bacterium that is widely used in dietary supplements and in milk and fermented dairy products (47). Originally a human intestinal isolate from the 1970s (8), this strain has since been examined extensively for various desirable traits. Due to the importance of understanding the molecular mechanisms involved in probiotic functions, the complete genome sequence of L. acidophilus NCFM was determined (2). The NCFM genome sequence serves as a blueprint for in silico identification of candidate gene loci and gene regulatory networks that may play essential roles in the survival and host interactions of this microorganism in the gastrointestinal (GI) tract, including genes involved in acid tolerance (5), bile tolerance (38, 42), adherence factors (20), environmental sensing and response (7), prebiotic sugar utilization (9), polysaccharide biosynthesis, oxalate degradation (6), and bacteriocin production (22). In addition, ongoing microarray gene expression studies have revealed specific gene sets of interest that are being investigated further. The fundamental approach for establishing functional roles for important gene features involves inactivation of the genes and subsequent phenotypic analyses of the associated isogenic mutants. Hence, an efficient gene knockout system is a valuable genetic tool for correlating key genotypes to phenotypes and for functional characterization of genes associated with probiotic attributes of L. acidophilus.The development of a site-directed lactococcal chromosomal integration system by Law and coworkers (34) involving the simultaneous use of a broad-host-range nonreplicative pWV01-derived vector (Ori⁺ RepA⁻) or so-called pORI-based vector and a temperature-sensitive helper plasmid, pVE6007 (35), that provides repA in trans for conditional replication of the pORI-based plasmids has greatly facilitated genetic studies of various gram-positive bacteria. This gene knockout strategy was adapted for use in L. acidophilus and L. gasseri with an alternate helper plasmid, pTRK669, that provides a higher permissive temperature range for thermophilic lactobacilli (46). This system has since been used successfully for generating numerous chromosomal insertion derivatives (1, 5, 9, 20, 23, 26, 38, 53). Nevertheless, the stability of the insertional mutations after single-crossover homologous recombination requires maintenance of antibiotic selection, and the same selection marker cannot be used to introduce multiple mutations into a strain. Furthermore, insertional inactivation of a specific target within an operon may have polar effects on downstream regions. These limitations were overcome by construction of markerless gene deletions via a double-crossover homologous recombination process involving plasmid integration and excision, where the wild-type allele is replaced with the mutant allele carrying an internal deletion in the target gene (6, 42). However, due to low efficiency of plasmid excision and allelic replacement and the lack of a selectable marker to detect these events, extensive screening is often necessary to isolate the desired recombinants.One practical approach to address this issue is to incorporate counterselectable genetic markers in allelic replacement systems to facilitate the recovery of plasmid-free derivatives following the second recombination event. Among the established counterselectable markers are the Bacillus subtilis sacB gene (encoding a levansucrase), which results in sucrose sensitivity (44), and the galK gene (encoding galactokinase), which mediates galactose or galactose analog toxicity (39, 49) in merodiploids that have not undergone the second homologous crossover event. Genes involved in purine and pyrimidine salvage pathways, specifically the genes coding for phosphoribosyltransferases (PRTases), such as upp (encoding uracil PRTase [UPRTase]), hprT (encoding hypoxanthine PRTase), pyrE/ura5 (encoding orotate PRTase), and pryF/ura3 (encoding orotidine-5′-phosphate decarboxylase), are also counterselectable markers that are widely used in bacterial, archaeal, and eukaryotic gene knockout systems (14, 15, 24, 25, 33, 41, 43, 54). These PRTases convert preformed purine and pyrimidine bases into corresponding nucleotide monophosphates for de novo nucleotide biosynthesis. The presence of a base analog that is also recognized as a substrate by the targeted PRTase can be lethal to cells when the base is incorporated into the nucleotide precursors, whereas a mutant with a nonfunctional PRTase is affected less by the toxicity of the base analog. The counterselection approach involved the use of an integration vector that provides ectopic expression of the PRTase-encoding gene in a PRTase-defective background host for gene-targeting deletions. Single-crossover integration of the recombinant plasmid is initially selected, and the recombination event renders the host sensitive to the PRTase-specific base analog due to expression of the plasmid-borne PRTase gene. Excision of the integrated plasmid following a second recombination event restores the base analog resistance phenotype of the host, which then serves as a counterselection strategy for rapid identification of plasmid-free recombinants that bear either the wild-type allele or the mutated allele of the targeted gene. The upp-based counterselection approach has previously been used for allelic replacement in B. subtilis and Enterococcus faecalis (24, 33).In the present study, we report the development of an improved markerless gene replacement system for L. acidophilus NCFM that involves the use of upp as a counterselectable marker for the existing pORI-based knockout system for positive selection of double recombinants. We demonstrated the efficiency of the upp counterselection scheme by deleting the slpX gene (LBA0512) encoding a novel secreted protein that was found to be associated with the S-layer complex in L. acidophilus. This study demonstrated the first functional counterselective gene replacement system for the genus Lactobacillus.     

The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite

We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular α-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin–antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as β-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria.