Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance

The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation. We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance. We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses. The position of the insertion of the transposon was located in 15 of these mutants. In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes. Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.     

Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.     

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase is involved in the tolerance of Arabidopsis seedlings to salt and osmotic stresses

In plants, pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) is a regulatory enzyme that participates in glycolysis and gluconeogenesis. Arabidopsis contains two PFPα subunit genes (PFPα1 and PFPα2) and two PFPβ subunit genes (PFPβ1 and PFPβ2). The single-knockout mutants of the PFP subunit genes were isolated, and double and quadruple pfp mutants were generated by crossing the single mutants. To elucidate the role of PFP in stress tolerance, the responses of the double and quadruple pfp knockout mutants to stress conditions, including osmotic and salt stresses, were examined. The seedling growth of the pfpα1/α2 and pfpβ1/β2 double mutants and the pfpα1/α2/β1/β2 quadruple mutant was severely retarded under salt and osmotic stress conditions compared with that of the wild type. The expression of PFP subunit genes increased in response to salt and osmotic stresses. In contrast, the vegetative growth of the wild type and pfp mutants after the seedling stage was similarly affected by salt and osmotic stresses. These findings suggest that PFP plays a role in the adaptation of Arabidopsis seedlings to salt and osmotic stresses.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.     

OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.     

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm² relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Identification of Genes Involved in Cytochrome c Biogenesis in Shewanella oneidensis, Using a Modified mariner Transposon

A modified mariner transposon, miniHimar RB1, was generated to mutagenize cells of the metal-reducing bacterium Shewanella oneidensis. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome c maturation genes ccmC and ccmF1, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome c production.     

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.Key words: Mesorhizobium loti, signature-tagged mutagenesis, mutant library, reverse genetics     

Isolation of mini-Tn5Km2 insertion mutants of Salmonella enterica serovar Oranienburg sensitive to NaCl-induced osmotic stress

We previously reported that the viability of Salmonella Oranienburg strains under NaCl stress was variable and depended on the strain's origin; food strains were resistant and patient strains sensitive to NaCl. Therefore, we mutagenized a food strain with a mini-Tn5Km2 transposon. Of 2,400 mutants screened, 15 NaCl-sensitive mutants were isolated, and 7 genes associated with NaCl-sensitivity were identified. The intact genes complemented their own food-strain mutants, but not patient-strain mutants, suggesting that the difference in NaCl-sensitivity between food and patient S. Oranienburg strains might not arise from a single gene mutation, but from change in multiple osmoregulatory mechanisms in Salmonella.     

Identification of genes required for growth under ethanol stress using transposon mutagenesis in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.     

Identification of genes involved in salt tolerance and symbiotic nitrogen fixation in chickpea rhizobium Mesorhizobium ciceri Ca181

In an attempt to find the genes involved in salt tolerance of the highly adaptable chickpea rhizobium strain, Mesorhizobium ciceri Ca181, a Tn5 transposon insertion library was generated and screened to identify five mutants with inability to survive in the presence of 0.1 M NaCl. The genes disrupted in these mutants due to insertion of the transposon were identified by sequencing of Tn5 flanking sequences after inverse PCR. One of the mutants had a disruption in diguanylate cyclase gene which is involved in bacterial biofilm formation and persistence. The second mutant had a disruption in an ABC transporter membrane protein gene, which is involved in the uptake of nutrients and cellular osmoprotection. The third mutant had a disruption in a gene showing homology with rhamnulose 1-phosphate aldolase which has an important role in the central metabolism of L-rhamnulose. The fourth mutant had a disruption in a capsule synthesis gene and the fifth mutant had an insertion in an oxidoreductase gene. When these mutants were inoculated into the host chickpea plant under normal non-saline conditions, they formed symbiotic nodules but with severely reduced nitrogenase activity. Hence, it appears that bacterial ability to adapt to hyper-osmotic salt stress conditions is also important for its nitrogen fixing ability in the chickpea root nodules. Allele mining for variant forms of the identified genes in the germplasm resources of M. ciceri may help in the development of highly adaptive and efficient nitrogen fixing strains of the chickpea rhizobium.     

Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α₂-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.