Functional haplodiploidy: a mechanism for the spread of insecticide resistance in an important international insect pest.

The coffee berry borer, Hypothenemus hampei, is the most important insect pest of coffee worldwide and has an unusual life history that ensures a high degree of inbreeding. Individual females lay a predominantly female brood within individual coffee berries and because males are flightless there is almost entirely full sib mating. We investigated the genetics associated with this interesting life history after the important discovery of resistance to the cyclodiene type insecticide endosulfan. Both the inheritance of the resistance phenotype and the resistance-associated point mutation in the gamma-aminobutyric acid receptor gene Rdl were examined. Consistent with haplodiploidy, males failed to express and transmit paternally derived resistance alleles. Furthermore, while cytological examination revealed that males are diploid, one set of chromosomes was condensed, and probably nonfunctional, in the somatic cells of all males examined. Moreover, although two sets of chromosomes were present in primary spermatocytes, the chromosomes failed to pair before the single meiotic division, and only one set was packaged in sperm. Thus, the coffee berry borer is "functionally" haplodiploid. Its genetics and life history may therefore represent an interesting intermediate step in the evolution of true haplodiploidy. The influence of this breeding system on the spread of insecticide resistance is discussed.     

Global extent of horizontal gene transfer

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. There have been many convincing evidences for HGT for specific genes or gene families, but there has been no estimate of the global extent of HGT. Here, we present a method of identifying HGT events within a given protein family and estimate the global extent of HGT in all curated protein domain families ( approximately 8,000) listed in the Pfam database. The results suggest four conclusions: (i) for all protein domain families in Pfam, the fixation of genes horizontally transferred is not a rampant phenomenon between organisms with substantial phylogenetic separations (1.1-9.7% of Pfam families surveyed at three taxonomic ranges studied show indication of HGT); (ii) however, at the level of domains, >50% of Archaea have one or more protein domains acquired by HGT, and nearly 30-50% of Bacteria did the same when examined at three taxonomic ranges. But, the equivalent value for Eukarya is <10%; (iii) HGT will have very little impact in the construction of organism phylogeny, when the construction methods use whole genomes, large numbers of common genes, or SSU rRNAs; and (iv) there appears to be no strong preference of HGT for protein families of particular cellular or molecular functions.     

The impact of long-distance horizontal gene transfer on prokaryotic genome size

Horizontal gene transfer (HGT) is one of the most dominant forces molding prokaryotic gene repertoires. These repertoires can be as small as ≈200 genes in intracellular organisms or as large as ≈9,000 genes in large, free-living bacteria. In this article we ask what is the impact of HGT from phylogenetically distant sources, relative to the size of the gene repertoire. Using different approaches for HGT detection and focusing on both cumulative and recent evolutionary histories, we find a surprising pattern of nonlinear enrichment of long-distance transfers in large genomes. Moreover, we find a strong positive correlation between the sizes of the donor and recipient genomes. Our results also show that distant horizontal transfers are biased toward those functional groups that are enriched in large genomes, showing that the trends in functional gene content and the impact of distant transfers are interdependent. These results highlight the intimate relationship between environmental and genomic complexity in microbes and suggest that an ecological, as opposed to phylogenetic, signal in gene content gains relative importance in large-genomed bacteria.     

Biased gene transfer mimics patterns created through shared ancestry

In phylogenetic reconstruction, two types of bacterial tyrosyl-tRNA synthetases (TyrRS) form distinct clades with many bacterial phyla represented in both clades. Very few taxa possess both forms, and maximum likelihood analysis of the distribution of TyrRS types suggests horizontal gene transfer (HGT), rather than an ancient duplication followed by differential gene loss, as the contributor to the evolutionary history of TyrRS in bacteria. However, for each TyrRS type, phylogenetic reconstruction yields phylogenies similar to the ribosomal phylogeny, revealing that frequent gene transfer has not destroyed the expected phylogeny; rather, the expected phylogenetic signal was reinforced or even created by HGT. We show that biased HGT can mimic patterns created through shared ancestry by in silico simulation. Furthermore, in cases where genomic synteny is sufficient to allow comparisons of relative gene positions, both tyrRS types occupy equivalent positions in closely related genomes, rejecting the loss hypothesis. Although the two types of bacterial TyrRS are only distantly related and only rarely coexist in a single genome, they have many features in common with alleles that are swapped between related lineages. We propose to label these functionally similar homologs as homeoalleles. We conclude that the observed phylogenetic pattern reflects both vertical inheritance and biased HGT and that the signal caused by common organismal descent is difficult to distinguish from the signal due to biased gene transfer.     

PCR法检测胃粘膜中的幽门螺杆菌

目的建立检测幽门螺杆菌（Ｈｐ）的更为敏感的聚合酶链反应（ＰＣＲ），并探讨它在Ｈｐ感染的诊断及根除效果判定中的应用．方法以一对合成的与Ｈｐ１６ＳｒＲＮＡ基因互补的寡核苷酸为引物（ＣＰ１／ＣＰ２），建立了从胃粘膜中检测Ｈｐ的ＰＣＲ反应，并与常规检测方法进行比较．结果ＰＣＲ方法检测Ｈｐ标准菌株及５０株临床分离菌株均产生５００ｂｐ片段，检出的最小ＤＮＡ量为０１ｐｇ，相当于１００个细菌细胞；所有１３株其它细菌及无Ｈｐ感染的人胃粘膜则无扩增产物出现．用该方法检测９６名初诊患者Ｈｐ感染情况及２１名药物治疗后患者Ｈｐ根除情况，并与胃粘膜活组织尿素酶试验、细菌培养及银染色方法比较，证实ＰＣＲ方法可以检出常规方法不能检出的少量Ｈｐ．结论ＰＣＲ是检测Ｈｐ的最为敏感的方法，有助于Ｈｐ感染的诊断和Ｈｐ根除的精确判断     

Horizontal gene transfer: a critical view

It has been suggested that horizontal gene transfer (HGT) is the "essence of phylogeny." In contrast, much data suggest that this is an exaggeration resulting in part from a reliance on inadequate methods to identify HGT events. In addition, the assumption that HGT is a ubiquitous influence throughout evolution is questionable. Instead, rampant global HGT is likely to have been relevant only to primitive genomes. In modern organisms we suggest that both the range and frequencies of HGT are constrained most often by selective barriers. As a consequence those HGT events that do occur most often have little influence on genome phylogeny. Although HGT does occur with important evolutionary consequences, classical Darwinian lineages seem to be the dominant mode of evolution for modern organisms.     

Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.

A nonvirulent Salmonella dublin flagellin-negative, aromatic-dependent live vaccine strain has been used to express hepatitis B virus surface antigen epitopes in an immunogenic form. The envelope proteins of the virion are encoded by the S gene, which contains the pre-S1, pre-S2, and S coding regions. Synthetic oligonucleotides corresponding to amino acid residues S-(122-137) and pre-S2-(120-145) were inserted in-frame into the hypervariable region of a cloned Salmonella flagellin gene, and the recombinant plasmids were introduced into a flagellin-negative aroA mutant live vaccine strain of S. dublin, SL5928. The flagellin gene was expressed in bacteria carrying the plasmids as detected by immunoblotting with anti-flagellin (H1-d) serum. Both the S and pre-S2 epitopes were detected in bacteria carrying the relevant plasmid by immunoblotting with anti-HBs (antibody to hepatitis B virus surface antigen) and anti-peptide antisera. Animals immunized intramuscularly or orally with the live recombinant bacteria developed antibodies specific to these hepatitis B virus epitopes as detected by ELISA.     

Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites

Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.     

Gorgeous mosaic of mitochondrial genes created by horizontal transfer and gene conversion

The best known outcome of horizontal gene transfer (HGT) is the introduction of novel genes, but other outcomes have been described. When a transferred gene has a homolog in the recipient genome, the native gene may be functionally replaced (and subsequently lost) or partially overwritten by gene conversion with transiently present foreign DNA. Here we report the discovery, in two lineages of plant mitochondrial genes, of novel gene combinations that arose by conversion between coresident native and foreign homologs. These lineages have undergone intricate conversion between native and foreign copies, with conversion occurring repeatedly and differentially over the course of speciation, leading to radiations of mosaic genes involved in respiration and intron splicing. Based on these findings, we develop a model--the duplicative HGT and differential gene conversion model--that integrates HGT and ongoing gene conversion in the context of speciation. Finally, we show that one of these HGT-driven gene-conversional radiations followed two additional types of conversional chimerism, namely, intramitochondrial retroprocessing and interorganellar gene conversion across the 2 billion year divide between mitochondria and chloroplasts. These findings expand our appreciation of HGT and gene conversion as creative evolutionary forces, establish plant mitochondria as a premiere system for studying the evolutionary dynamics of HGT and its genetic reverberations, and recommend careful examination of bacterial and other genomes for similar, likely overlooked phenomena.     

Dynamic colonization of Helicobacter pylori in human gastric mucosa

OBJECTIVE: To investigate the dynamic behaviour of Helicobacter pylori in the colonization of the human gastric mucosa in patients previously treated for H. pylori infection. MATERIAL AND METHODS: Twenty-one dyspeptic patients were included in the study. Biopsies from each individual were taken and analysed for H. pylori detection using cultural, molecular and ultrastructural methods. RESULTS: Through culture, H. pylori was isolated in 7 out of 21 patients and the detection of the minimum inhibitory concentration (MIC) against drugs commonly used in H. pylori therapy revealed a susceptibility panel in which only one strain was multidrug resistant. By studying the expression of the H. pylori glmM constitutive gene, viable H. pylori cells were detected in 19 out of 21 analysed biopsies. In these positive cases, the expression of the Quorum-Sensing related gene, luxS, was always detected. The analysis of glmM and luxS sequences confirmed the H. pylori identity. Scanning electron microscopy (SEM) analysis of biopsies from patients harbouring culturable bacteria showed a prevalent "S-shape" H. pylori morphotype co-existent with coccoid aggregated bacteria embedded in an abundant matrix; while samples from patients shown as H. pylori-positive only through the molecular method showed clustered coccoid bacteria arranged in a microbial biofilm. CONCLUSIONS: In the present work we describe a new scenario in H. pylori mucosa colonization suggesting, in infection recalcitrance, the planning of more efficacious protocols in order also to identify camouflaged and protected clustered bacteria, taking into account this serious microbial problem in medicine in the recommendation of therapeutic regimens.     

Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas

Genomic islands (GEI) comprise a recently recognized large family of potentially mobile DNA elements and play an important role in the rapid differentiation and adaptation of bacteria. Most importantly, GEIs have been implicated in the acquisition of virulence factors, antibiotic resistances or toxic compound metabolism. Despite detailed information on coding capacities of GEIs, little is known about the regulatory decisions in individual cells controlling GEI transfer. Here, we show how self-transfer of ICEclc, a GEI in Pseudomonas knackmussii B13 is controlled by a series of stochastic processes, the result of which is that only a few percent of cells in a population will excise ICEclc and launch transfer. Stochastic processes have been implicated before in producing bistable phenotypic transitions, such as sporulation and competence development, but never before in horizontal gene transfer (HGT). Bistability is instigated during stationary phase at the level of expression of an activator protein InrR that lays encoded on ICEclc, and then faithfully propagated to a bistable expression of the IntB13 integrase, the enzyme responsible for excision and integration of the ICEclc. Our results demonstrate how GEI of a very widespread family are likely to control their transfer rates. Furthermore, they help to explain why HGT is typically confined to few members within a population of cells. The finding that, despite apparent stochasticity, HGT rates can be modulated by external environmental conditions provides an explanation as to why selective conditions can promote DNA exchange.     

Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes

Horizontal gene transfer (HGT) can radically alter the genomes of microorganisms, providing the capacity to adapt to new lifestyles, environments, and hosts. However, the extent of HGT between eukaryotes is unclear. Using whole-genome, gene-by-gene phylogenetic analysis we demonstrate an extensive pattern of cross-kingdom HGT between fungi and oomycetes. Comparative genomics, including the de novo genome sequence of Hyphochytrium catenoides, a free-living sister of the oomycetes, shows that these transfers largely converge within the radiation of oomycetes that colonize plant tissues. The repertoire of HGTs includes a large number of putatively secreted proteins; for example, 7.6% of the secreted proteome of the sudden oak death parasite Phytophthora ramorum has been acquired from fungi by HGT. Transfers include gene products with the capacity to break down plant cell walls and acquire sugars, nucleic acids, nitrogen, and phosphate sources from the environment. Predicted HGTs also include proteins implicated in resisting plant defense mechanisms and effector proteins for attacking plant cells. These data are consistent with the hypothesis that some oomycetes became successful plant parasites by multiple acquisitions of genes from fungi.     

糖尿病肾病小鼠新相关基因表达载体的构建及其在大肠杆菌中的表达

目的：构建小鼠“REKEN cDNA 0610006H10”基因表达载体pPRO-6AB，了解此表达载体在大肠杆菌中表达的情况。方法：从克隆有“REKEN cDNA 0610006H10”基因cDNA的载体pUCm-6AB中切取长约1．1kb的“REKENcDNA 0610006H10”基因cDNA，将它克隆到原核表达载体pPROTet-E的多克隆位点中。以限制性内切酶酶切分析的方法对重组克隆进行初步鉴定，以测序法对重组子进行验证。在确证得到了预期的表达载体pPRO-6AB后，以氯化钙转染法将表达载体导人大肠杆菌BL21PRO中作蛋白表达分析。蛋白分析方法采用SDS变性聚丙烯酰胺凝胶电泳，所用胶浓度为10％，交联度为3．3％。电泳结束后利用考马丝亮蓝染色法进行染色。结果：得到了预期的“REKEN cDNA 0610006H10”基因原核表达载体pPRO-6AB“REKEN cDNA 0610006H10”基因成功地在大肠杆菌中实现了表达。结论：我们成功地构建了小鼠“REKEN cDNA 0610006H10”基因表达载体pPRO-6AB，在大肠杆菌中成功地进行了“REKEN cDNA 0610006H10”基因的表达，从而为进一步研究“REKEN cDNA 0610006H10”基因编码蛋白的结构和功能，探讨“REKEN cDNA 0610006H10”基因在糖尿病肾病发生、发展过程中的可能作用创造了条件。     

Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women

ABSTRACT: BACKGROUND: The presence of hydrogen peroxide (H2O2) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H2O2- producers. METHODS: Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR. RESULTS: Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45). CONCLUSIONS: Among HIV-1 seropositive women, there was a moderate level of concordance between of H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.     

Bacteriophage f1 infection of Escherichia coli: identification and possible processing of f1-specific mRNAs in vivo.

[3H]Uracil-pulse-labeled RNA from Escherichia coli infected with f1 bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 41 degrees C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein. The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition to rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phage-specific proteins.     

Anaphylaxis associated with the ingestion of Goji berries (Lycium barbarum)

Goji berry (wolfberry), a member of the Solanacea family, has been recently introduced in Western countries and its consumption has increased rapidly. The objectives of the study were to describe the cases of 2 patients who experienced allergic symptoms after Goji berry consumption, to identify the protein profile of the extract, to analyze the allergenic profile of individuals, and to determine cross-reactivity with other members of the Solanaceae family (tomato). We describe 2 cases of allergic reaction, 1 of which was an anaphylactic reaction, after Goji berry ingestion. A Goji berry extract was manufactured and immunochemically characterized. The patients were skin prick tested with a battery of common aeroallergens including mites, epithelia, and molds. Individuals were also skin prick tested with food allergens, including Goji berries. A positive skin prick test and specific immunoglobulin (Ig) E to Goji berry was detected in both cases. Serum samples recognized a 9-kDa band, probably related to lipid transfer proteins (LTPs). Cross-reactivity with tomato was analyzed by inhibition studies, which showed that the 9-kDa band was totally inhibited by the tomato extract. This study describes the first 2 cases of allergic reaction following Goji berry ingestion. LTPs seem to be involved in allergic sensitization to Goji berries, as evidenced by cross-reactivity with tomato.     

Human seminal relaxin is a product of the same gene as human luteal relaxin.

Unlike that of other species, which have only one gene encoding relaxin, the human genome contains two nonallelic genes for relaxin, designated H1 and H2, which encode markedly different relaxin peptides. Whereas human relaxin gene H2 is selectively expressed in the ovary, no ovarian expression of gene H1 has been detected. Since relaxin is actively produced in the human male, it is possible to postulate divergent gene expression of relaxin in the male and female. We examined this question directly through the structural determination of human seminal relaxin and its comparison with the structure of human luteal relaxin. Partially purified relaxin, prepared from pooled human seminal plasma which had been delipidated by extraction with acid acetone and hexane, subjected to two cycles of HPLC and an additional purification step by ion-exchange chromatography, was further purified by immunoaffinity chromatography, using a monoclonal antibody to the H2 relaxin A chain which cross-reacts with synthetic H1 relaxin, followed by an additional HPLC step performed on a C4 reverse-phase column. The recovered, purified relaxin was then analyzed by N-terminal gas-phase sequencing and fast atom bombardment mass spectroscopy for determination of the amino acid sequence and molecular ions of the A and B chains, respectively. The results demonstrate that the structure of the predominant relaxin in human semen plasma is derived from the product of the H2 gene, consisting of a N-terminal pyroglutamic acid A-24 A chain and a mixture of B-26 and B-27 B chains. With the exception of degradation of the seminal relaxin B chain C-terminus, this structure is identical to the structure of human luteal relaxin. Therefore, both human seminal and luteal relaxin are products of the H2 gene.     

Relationship of smoking and coffee and alcohol consumption with seroconversion to Helicobacter pylori: a longitudinal study in hospital workers

BACKGROUND AND AIM: There are few data concerning the relationship between Helicobacter pylori seroconversion, and smoking habits and coffee and alcohol consumption. The aim of the present study was to investigate the relationship between smoking habits, coffee and alcohol consumption, and H. pylori seroconversion. METHODS: The data used were derived from a sample of 238 subjects (hospital employees) who were initially (on 1994) seronegative to H. pylori. These subjects were tested again 5 years later (1999). Information concerning smoking habits and coffee and alcohol consumption was collected by the use of a special questionnaire, which was completed by the same cohort of subjects in two different periods (1994 and 1999). Logistic regression was used to assess the relationship between H. pylori seroconversion and the aforementioned lifestyle factors. RESULTS: Neither smoking nor coffee consumption was significantly related to H. pylori seroconversion. Adjusted odds ratio for alcohol drinkers as compared with non-/occasional drinkers was 0.59 (95% confidence interval [CI]: 0.31-1.16, P = 0.13). However, the odds ratio was significantly lower (0.26, 95%CI: 0.07-0.95, P = 0.042) in subjects who reported moderate alcohol consumption at first (1994) examination, as compared with non-/occasional drinkers. Small and heavy drinking were not associated with H. pylori seroconversion. CONCLUSIONS: There is no significant relation between H. pylori seroconversion and smoking and coffee consumption. The present findings suggest that moderate alcohol consumption might be inversely associated with H. pylori seroconversion.     

Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of ~100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.