Marker Exchange Research Articles

Integrated conjugative plasmid drives high frequency chromosomal gene transfer in Sulfolobus islandicus.

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance from pM164. We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host-encoded marker exchange (ME) mechanism.

Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties.

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato‐growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid‐cured strains found that Cm‐mediated hypersensitive response (HR) in the Cm‐resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI‐encoded serine proteases into Cm∆PI identified that the HR is elicited by the protease ChpG. Eggplant leaves infiltrated with a chpG marker exchange mutant (CmΩchpG) did not display an HR, and infiltration of purified ChpG protein elicited immune responses in eggplant but not in Cm‐susceptible tomato. Virulence assays found that while wild‐type Cm and the CmΩchpG complemented strain were nonpathogenic on eggplant, CmΩchpG caused wilt and canker symptoms. Additionally, bacterial populations in CmΩchpG‐inoculated eggplant stem tissues were c.1000‐fold higher than wild‐type and CmΩchpG‐complemented Cm strains. Pathogenicity tests conducted in multiple Cm‐resistance eggplant varieties demonstrated that immunity to Cm is dependent on ChpG in all tested varieties, indicating that ChpG‐recognition is conserved in eggplant. ChpG‐mediated avirulence interactions were disabled by alanine substitution of serine231 of the serine protease catalytic triad, suggesting that protease activity is required for immune recognition of ChpG. Our study identified ChpG as a novel avirulence protein that is recognized in resistant eggplant varieties and restricts the host range of Cm.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

ABSTRACTThe ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth’s biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2.IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria.

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.

Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate production by a prpC mutant of Herbaspirillum seropedicae Z69

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters produced by a wide range of bacteria as carbon and energy reserves. PHA accumulation is typically increased under unbalanced growth conditions and with carbon source in excess. Although polyhydroxybutyrate (PHB) could be used for specific applications, it is brittle and not a useful alternative for plastics like polypropylene. Far more useful polypropylene-like PHAs, are copolymers composed of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Propionic acid is one of the carbon sources that can be used to generate 3HV. A mutant derived from Herbaspirillum seropedicae Z69, a strain previously described as capable of producing P(3HB-co-3HV) from propionic acid, was constructed to increase 3HV biosynthetic efficiency. The strategy involved elimination of a catabolic route for propionyl-CoA by deficiency marker exchange of a selected gene. The mutant (Z69Prp) was constructed by elimination of the 2-methylcitrate synthase (PrpC) gene of the 2-methylcitrate cycle for propionate catabolism. Strain Z69Prp was unable to grow on sodium propionate, but in cultures with glucose-propionate accumulated 50% of its dry weight as copolymer. Z69Prp had 14.1 mol% 3HV; greater than that of strain Z69 (2.89 mol%). The 3HV yield from propionic acid (Y3HV/prop) was 0.80 g g−1, and below the maximum theoretical value (1.35 g g−1).

A novel bacterial sulfur oxidation pathway provides a new link between the cycles of organic and inorganic sulfur compounds.

Dimethylsulfide (DMS) plays a globally significant role in carbon and sulfur cycling and impacts Earth's climate because its oxidation products serve as nuclei for cloud formation. While the initial steps of aerobic DMS degradation and the fate of its carbon atoms are reasonably well documented, oxidation of the contained sulfur is largely unexplored. Here, we identified a novel pathway of sulfur compound oxidation in the ubiquitously occurring DMS-degrader Hyphomicrobium denitrificans XT that links the oxidation of the volatile organosulfur compound with that of the inorganic sulfur compound thiosulfate. DMS is first transformed to methanethiol from which sulfide is released and fully oxidized to sulfate. Comparative proteomics indicated thiosulfate as an intermediate of this pathway and pointed at a heterodisulfide reductase (Hdr)-like system acting as a sulfur-oxidizing entity. Indeed, marker exchange mutagenesis of hdr-like genes disrupted the ability of H. denitrificans to metabolize DMS and also prevented formation of sulfate from thiosulfate provided as an additional electron source during chemoorganoheterotrophic growth. Complementation with the hdr-like genes under a constitutive promoter rescued the phenotype on thiosulfate as well as on DMS. The production of sulfate from an organosulfur precursor via the Hdr-like system is previously undocumented and provides a new shunt in the biogeochemical sulfur cycle. Furthermore, our findings fill a long-standing knowledge gap in microbial dissimilatory sulfur metabolism because the Hdr-like pathway is abundant not only in chemoheterotrophs, but also in a wide range of chemo- and photolithoautotrophic sulfur oxidizers acting as key players in global sulfur cycling.

Application of the Cre/lox System to Construct Auxotrophic Markers for Quantitative Genetic Analyses in Fusarium graminearum.

The bacteriophage P1 Cre/lox system has been utilized in diverse fungi for marker recycling and exchange, generation of targeted chromosome translocations, and targeted deletion of interstitial chromosome segments. Here we show the application of this tool in the wheat and maize pathogen, Fusarium graminearum. We explored three different ways to introduce Cre into strains with floxed genes, namely transformation with an episomal or integrative plasmid (pLC28), fusion of protoplasts of strains carrying floxed genes with strains expressing Cre by forcing heterokaryons, and crosses between strains with floxed genes and strains expressing Cre to isolate progeny in which the target genes had been deleted during the cross. We used this system for the construction of strains bearing auxotrophic markers that were generated by gene replacement with positively selectable markers followed by Cre-mediated marker excision. In addition, updated protocols for transformation and crosses for F. graminearum are provided. In combination, strains and tools developed here add to the arsenal of methods that can be used to carry out molecular genetics with F. graminearum.

Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System.

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoidtetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoidtetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b.

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm(r) mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm(r) mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

New cassettes for single-step drug resistance and prototrophic marker switching in fission yeast.

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein-protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4(+) gene or the G418-resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker, or to completely reconstruct a particular strain with a different marker, single-step exchange protocols of markers are a time-saving alternative. In recent years, dominant drug resistance markers (DDRMs) against clonNAT, hygromycin B and bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes, natMX, hphMX and bleMX, carry the TEF promotor and terminator sequences from Ashbya gossypii as kanMX; this provides flanking homologies to enable single-step marker swapping by homologous gene targeting. To expand this very useful toolset for single-step marker exchange, I constructed MX cassettes containing the nutritional markers arg3(+), his3(+), leu1(+) and ura4(+). Furthermore, a set of constructs was created to enable single-step exchange of ura4(+) to kanMX6, natMX4 and hphMX4. The functionality of the cassettes is demonstrated by successful single-step marker swapping at several loci. These constructs allow straightforward and rapid remarking of existing ura4(+) - and MX-deleted and -tagged strains.

A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans.

The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor.

An effector gene hopA1 influences on virulence, host specificity, and lifestyles of Pseudomonas cichorii JBC1

Pseudomonas cichorii is a devastating pathogen which infects a wide range of ornamental as well as agricultural crops worldwide. Characterization of virulence genes helps to understand pathogens' infection processes, which may lead to development of resistant crops. For functional validation of novel genes, we re-constructed pUCP18 vector with λ phage red operon and sacB gene (pUCP18_RedS), which simplified conventional marker exchange system. The effector gene hopA1 of P. cichorii JBC1 was marker exchanged with PCR product of kanamycin gene flanked by hopA1 flanking region using pUCP18_RedS. The virulence and internal growth of hopA1 defective mutant (ΔhopA1) in tomato seedlings was significantly reduced compared to wild type (WT) and hopA1 complemented strain (ΔhopA1::phopA1). The analysis on role of hopA1 in host range revealed that P. cichorii was hopA1-dependent to infect cabbage, tomato, soybean, hot pepper, and cucumber, but not melon and eggplant. Despite the similarity in growth pattern, the biofilm formation and swarming motility of ΔhopA1 were significantly reduced compared to WT and ΔhopA1::phopA1. The results of this study indicate that hopA1 plays a significant role not only in virulence and host specificity, but also motility and biofilm formation of P. cichorii which may influence the infection processes.

GltB/D Mutants of Xanthomonas oryzae pv. oryzae are Virulence Deficient

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Upon clip inoculation of rice leaves, Xoo causes typical V-shaped lesions whose leading edge moves through the mid-veinal region. We have isolated a virulence deficient mutant of Xoo, referred to as BXO808 that causes limited lesions which primarily extend through the side-veinal regions of rice leaves. Functional complementation studies identified a clone, pSR19, from a cosmid genomic library that restored wild-type virulence and lesion phenotype to BXO808. Transposon mutagenesis of the pSR19 clone, marker exchange experiments, and targeted mutagenesis, revealed that the BXO808 phenotype is due to mutation in the gltB/D genes of Xoo, which encode glutamate synthase subunits α and β, respectively. The gltB/D mutants that were generated in this study also exhibited virulence deficiency, an altered lesion phenotype and growth deficiency on minimal medium with low levels of ammonium as a sole nitrogen source. This is the first report that mutations in the gltB/D genes of Xoo cause virulence deficiency.

HrcQ is necessary for Xanthomonas oryzae pv. oryzae HR-induction in non-host tobacco and pathogenicity in host rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most destructive diseases of rice (Oryza sativa L.) worldwide. The type III secretion system (T3SS) of Xoo, encoded by the hrp (hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response (HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99A, by leaf-cutting inoculation. Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192–70,201 in the genome of PXO99A, disrupting the type III hrc (hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99A mutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa

Background Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.Methods and Principal FindingsTo facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.ConclusionsWe have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Disruption of rsmA gene of Pectobacterium carotovorum subsp. carotovorum LY34 and effect on pathogenicity

The rsmA gene was cloned from soft-rot bacterium Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34), and its role in pathogenicity was investigated by marker exchange mutagenesis. From a cosmid library of Pcc LY34 genomic DNA, a positive clone carrying the rsmA gene was selected, and the gene was cloned by polymerase chain reaction (PCR) amplification. The gene is 186 bp in size and encodes a protein of 62 amino acids with a predicted molecular mass of 6,839 Da. The calculated pI of the RsmA is 8.16. The phylogenetic tree showed that the RsmA of Pcc LY34 appeared genetically identical to the CsrA of Pectobacterium atrosepticum SCRI1043 (100% identity) and similar to the CsrA of Yersinia pestis KIM10+ (98.3%). The gene was disrupted by the Kmr gene, and the cells became mutated (i.e., RsmA− mutant). The pathogenicity test revealed that the disease rating of the RsmA− mutant only differed slightly from that of the wild type on a slice of potato tuber and a Chinese cabbage stalk. These results suggest that RsmA is not an essential factor for the pathogenicity of Pcc LY34 and that the rsmA gene of Pcc LY34 is not completely derepressed in the RsmA− mutant for virulence-related genes, contrary to the results of Erwinia carotovora subsp. carotovora RsmA− mutant, which proved hypervirulent for celery petioles. These results showed that the microenvironmental conditions of the host and/or strain of pathogen are important for the coordination of virulence gene expression.

AtaA, a New Member of the Trimeric Autotransporter Adhesins from Acinetobacter sp. Tol 5 Mediating High Adhesiveness to Various Abiotic Surfaces

Acinetobacter sp. Tol 5 exhibits an autoagglutinating nature and noteworthy adhesiveness to various abiotic surfaces from hydrophobic plastics to hydrophilic glass and stainless steel. Although previous studies have suggested that bacterionanofibers on Tol 5 cells are involved in the adhesive phenotype of Tol 5, the fiber that directly mediates Tol 5 adhesion has remained unknown. Here, we present a new member of trimeric autotransporter adhesins designated AtaA, which we discovered by analyzing a less adhesive mutant of Tol 5, T1, obtained by transposon mutagenesis. AtaA forms thinner and shorter nanofibers than fimbriae on Tol 5 cells. We performed target disruption of ataA by allelic marker exchange, and the resulting ΔataA strain was complemented with ataA on the Escherichia coli-Acinetobacter shuttle vector, which was newly constructed. These results proved that AtaA is essential for Tol 5’s autoagglutinating nature and high adhesiveness to surfaces of various materials. In addition, the adhesiveness to solid surfaces mediated by AtaA is notably higher than that mediated by YadA of Yersinia enterocolitica WA-314. Moreover, and importantly, these characteristics can be conferred to the non-adhesive, non-agglutinating bacterium Acinetobacter sp. ADP1 in trans by transformation with ataA, with expected applications to microbial immobilization.

Membrane and cytoplasmic marker exchange between malignant neoplastic cells and fibroblasts via intermittent contact: increased tumour cell diversity independent of genetic change

We previously demonstrated that human osteosarcoma cells (SAOS-2) induce contact-dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h-GF) using SAOS-2 alkaline phosphatase (AP) to identify cells. However, h-GF apoptosis did not occur, despite reduction in AP-negative h-GF number (p < 0.01) and enhancement of this by h-GF TNFα pretreatment (p < 0.01). We suggest that TNFα-enhanced transfer of membrane AP from SAOS-2 to h-GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co-cultures of membrane-labelled h-GF (marker-DiO) and SAOS-2 (marker-DiD) generated dual-labelled cells, primarily at the expense of single labelled h-GF (p < 0.001), suggesting predominant membrane transfer from SAOS-2 to h-GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS-2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h-GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h-GF prelabelled with cytoplasmic DDAO-SE and nuclear Syto59, co-cultured with SAOS-2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h-GF to SAOS-2 (p < 0.05). Pretreating h-GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual-labelled cells had a morphological phenotype differing from SAOS-2 and h-GF (p < 0.001). Time-lapse microscopy revealed extensive migration of SAOS-2 and cell process contact with h-GF, with the appearance of SAOS-2 indulging in 'cellular sipping' from h-GF. Similar exchange of membrane was seen between h-GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200-B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression.

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.