Journal articles: 'Bacterial complementation'

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Fröderberg, Linda, Thomas Röhl, Klaas-Jan van Wijk, and Jan-Willem L. de Gier. "Complementation of bacterial SecE by a chloroplastic homologue." FEBS Letters 498, no. 1 (May 30, 2001): 52–56. http://dx.doi.org/10.1016/s0014-5793(01)02494-2.

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Crépin, Sébastien, Josée Harel, and Charles M. Dozois. "Chromosomal Complementation Using Tn7Transposon Vectors in Enterobacteriaceae." Applied and Environmental Microbiology 78, no. 17 (June 15, 2012): 6001–8. http://dx.doi.org/10.1128/aem.00986-12.

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ABSTRACTGenetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn7transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn7systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn7and transposase vectors to provide a more versatile system. Transposition of Tn7at the chromosomalattTn7site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn7vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.

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Merkel, Susan, Adam Parks, and Buck Hanson. "A Small Group Activity About Bacterial Regulation And Complementation †." Journal of Microbiology & Biology Education 11, no. 2 (December 20, 2010): 152–55. http://dx.doi.org/10.1128/jmbe.v11i2.196.

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Cahoon, Laty A., and Nancy E. Freitag. "Identification of Conserved and Species-Specific Functions of the Listeria monocytogenes PrsA2 Secretion Chaperone." Infection and Immunity 83, no. 10 (July 27, 2015): 4028–41. http://dx.doi.org/10.1128/iai.00504-15.

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The Gram-positive bacteriumListeria monocytogenesis a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolylcis/transisomerase and chaperone that is dispensable for bacterial growth in broth culture but essential forL. monocytogenesvirulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation ofL. monocytogenesmutants lackingprsA2. PrsA homologues encoded byBacillus subtilis,Streptococcus pyogenes,Streptococcus pneumoniae,Streptococcus mutans,Staphylococcus aureus, andLactococcus lactiswere examined for functional complementation of a variety ofL. monocytogenesPrsA2-associated phenotypes central toL. monocytogenespathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity forL. monocytogenestarget proteins required for pathogenesis. TheL. monocytogenesPrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation.

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Pribat, Anne, Linda Jeanguenin, Aurora Lara-Núñez, Michael J. Ziemak, John E. Hyde, Valérie de Crécy-Lagard, and Andrew D. Hanson. "6-Pyruvoyltetrahydropterin Synthase Paralogs Replace the Folate Synthesis Enzyme Dihydroneopterin Aldolase in Diverse Bacteria." Journal of Bacteriology 191, no. 13 (April 24, 2009): 4158–65. http://dx.doi.org/10.1128/jb.00416-09.

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ABSTRACT Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.

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Gottier, Petra, Mauro Serricchio, Rita Vitale, Angela Corcelli, and Peter Buetikofer. "Cross-species complementation of bacterial- and eukaryotic-type cardiolipin synthases." Microbial Cell 4, no. 11 (November 6, 2017): 376–83. http://dx.doi.org/10.15698/mic2017.11.598.

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Kraemer, Susanne A., and Gregory J. Velicer. "Social complementation and growth advantages promote socially defective bacterial isolates." Proceedings of the Royal Society B: Biological Sciences 281, no. 1781 (April 22, 2014): 20140036. http://dx.doi.org/10.1098/rspb.2014.0036.

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Social interactions among diverse individuals that encounter one another in nature have often been studied among animals but rarely among microbes. For example, the evolutionary forces that determine natural frequencies of bacteria that express cooperative behaviours at low levels remain poorly understood. Natural isolates of the soil bacterium Myxococcus xanthus sampled from the same fruiting body often vary in social phenotypes, such as group swarming and multicellular development. Here, we tested whether genotypes highly proficient at swarming or development might promote the persistence of less socially proficient genotypes from the same fruiting body. Fast-swarming strains complemented slower isolates, allowing the latter to keep pace with faster strains in mixed groups. During development, one low-sporulating strain was antagonized by high sporulators, whereas others with severe developmental defects had those defects partially complemented by high-sporulating strains. Despite declining in frequency overall during competition experiments spanning multiple cycles of development, developmentally defective strains exhibited advantages during the growth phases of competitions. These results suggest that microbes with low-sociality phenotypes often benefit from interacting with more socially proficient strains. Such complementation may combine with advantages at other traits to increase equilibrium frequencies of low-sociality genotypes in natural populations.

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Valencia-Burton, Maria, Ron M. McCullough, Charles R. Cantor, and Natalia E. Broude. "RNA visualization in live bacterial cells using fluorescent protein complementation." Nature Methods 4, no. 5 (April 1, 2007): 421–27. http://dx.doi.org/10.1038/nmeth1023.

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Green, Michael R., and Joseph Sambrook. "Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation." Cold Spring Harbor Protocols 2019, no. 12 (December 2019): pdb.prot101329. http://dx.doi.org/10.1101/pdb.prot101329.

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Sambrook, Joseph, and David W. Russell. "Screening Bacterial Colonies Using X-gal and IPTG: α-Complementation." Cold Spring Harbor Protocols 2006, no. 1 (June 2006): pdb.prot3945. http://dx.doi.org/10.1101/pdb.prot3945.

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Canyuk, B., S. P. Craig III., and A. E. Eakin. "Bacterial complementation as a means to test enzyme-ligand interactions." Applied Microbiology and Biotechnology 50, no. 2 (August 27, 1998): 181–86. http://dx.doi.org/10.1007/s002530051274.

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Entcheva, Plamena, Donald A. Phillips, and Wolfgang R. Streit. "Functional Analysis of Sinorhizobium meliloti Genes Involved in Biotin Synthesis and Transport." Applied and Environmental Microbiology 68, no. 6 (June 2002): 2843–48. http://dx.doi.org/10.1128/aem.68.6.2843-2848.2002.

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ABSTRACT External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.

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Lopes Ferreira, Nicolas, and Jean-Hervé Alix. "The DnaK Chaperone Is Necessary for α-Complementation of β-Galactosidase in Escherichia coli." Journal of Bacteriology 184, no. 24 (December 15, 2002): 7047–54. http://dx.doi.org/10.1128/jb.184.24.7047-7054.2002.

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ABSTRACT We show here the involvement of the molecular chaperone DnaK from Escherichia coli in the in vivo α-complementation of the β-galactosidase. In the dnaK756(Ts) mutant, α-complementation occurs when the organisms are grown at 30°C but not at 37 or 40°C, although these temperatures are permissive for bacterial growth. Plasmid-driven expression of wild-type dnaK restores the α-complementation in the mutant but also stimulates it in a dnaK + strain. In a mutant which contains a disrupted dnaK gene (ΔdnaK52::Cmr), α-complementation is also impaired, even at 30°C. This observation provides an easy and original phenotype to detect subtle functional changes in a protein such as the DnaK756 chaperone, within the physiologically relevant temperature.

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Liu, Hong-he, Ke-wei Zheng, Yi-de He, Quan Chen, Yu-hua Hao, and Zheng Tan. "RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation." Chemical Science 7, no. 7 (2016): 4573–81. http://dx.doi.org/10.1039/c5sc03946k.

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15

Rubin, Jaime S., and Gordon F. Whitmore. "Complementation of DNA-repair deficiencies in Chinese hamster ovary cells." Biochemistry and Cell Biology 65, no. 9 (September 1, 1987): 803–10. http://dx.doi.org/10.1139/o87-105.

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Genetic mapping studies in bacterial, lower eukaryotic, and mammalian systems have demonstrated that the enzyme or enzyme complex involved in the initial incision step of the DNA excision repair pathway is coded for by more than one genetic locus. This paper reports the results of complementation studies that were performed with a number of DNA repair deficient Chinese hamster ovary cell lines. Complementation abilities were measured by comparing the survival of selected mutant × mutant hybrids with that of the tetrapoloid wild type after exposure to a number of physical and chemical agents. In all cases studied, hybrids formed from two different mutant lines showed resistance similar to that of the wild-type line. These results not only demonstrate that the mutant lines were in different complementation groups, but that complementation of a DNA-repair defect associated with one particular agent can complement the defect for another DNA-damaging agent.

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Parker, Richard P., Alison D. Walters, and James P. J. Chong. "Bacterial and eukaryotic systems collide in the three Rs of Methanococcus." Biochemical Society Transactions 39, no. 1 (January 19, 2011): 111–15. http://dx.doi.org/10.1042/bst0390111.

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Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.

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Ulitzur, Nirit, and Shimon Ulitzur. "New Rapid and Simple Methods for Detection of Bacteria and Determination of Their Antibiotic Susceptibility by Using Phage Mutants." Applied and Environmental Microbiology 72, no. 12 (September 22, 2006): 7455–59. http://dx.doi.org/10.1128/aem.00761-06.

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ABSTRACT Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria (“infectious centers”). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.

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Kitabatake, Makoto, Man Wah So, Debra L. Tumbula, and Dieter Söll. "Cysteine Biosynthesis Pathway in the ArchaeonMethanosarcina barkeri Encoded by Acquired Bacterial Genes?" Journal of Bacteriology 182, no. 1 (January 1, 2000): 143–45. http://dx.doi.org/10.1128/jb.182.1.143-145.2000.

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ABSTRACT The pathway of cysteine biosynthesis in archaea is still unexplored. Complementation of a cysteine auxotrophic Escherichia coli strain NK3 led to the isolation of the Methanosarcina barkeri cysK gene [encoding O-acetylserine (thiol)-lyase-A], which displays great similarity to bacterialcysK genes. Adjacent to cysK is an open reading frame orthologous to bacterial cysE (serine transacetylase) genes. These two genes could account for cysteine biosynthesis in this archaeon. Analysis of recent genome data revealed the presence of bacteria-like cysM genes [encodingO-acetylserine (thiol)-lyase-B] in Pyrococcusspp., Sulfolobus solfataricus, and Thermoplasma acidophilum. However, no orthologs for these genes can be found in Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidus, implying the existence of unrecognizable genes for the same function or a different cysteine biosynthesis pathway.

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Uroz, S., C. Calvaruso, M. P. Turpault, J. C. Pierrat, C. Mustin, and P. Frey-Klett. "Effect of the Mycorrhizosphere on the Genotypic and Metabolic Diversity of the Bacterial Communities Involved in Mineral Weathering in a Forest Soil." Applied and Environmental Microbiology 73, no. 9 (March 9, 2007): 3019–27. http://dx.doi.org/10.1128/aem.00121-07.

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ABSTRACT To date, several bacterial species have been described as mineral-weathering agents which improve plant nutrition and growth. However, the possible relationships between mineral-weathering potential, taxonomic identity, and metabolic ability have not been investigated thus far. In this study, we characterized a collection of 61 bacterial strains isolated from Scleroderma citrinum mycorrhizae, the mycorrhizosphere, and the adjacent bulk soil in an oak forest. The ability of bacteria to weather biotite was assessed with a new microplate bioassay that measures the pH and the quantity of iron released from this mineral. We showed that weathering bacteria occurred more frequently in the vicinity of S. citrinum than in the bulk soil. Moreover, the weathering efficacy of the mycorrhizosphere bacterial isolates was significantly greater than that of the bulk soil isolates. All the bacterial isolates were identified by partial 16S rRNA gene sequence analysis as members of the genera Burkholderia, Collimonas, Pseudomonas, and Sphingomonas, and their carbon metabolism was characterized by the BIOLOG method. The most efficient isolates belonged to the genera Burkholderia and Collimonas. Multivariate analysis resulted in identification of three metabolic groups, one of which contained mainly bacterial isolates associated with S. citrinum and exhibiting high mineral-weathering potential. Therefore, our results support the hypothesis that by its carbon metabolism this fungus selects in the bulk soil reservoir a bacterial community with high weathering potential, and they also address the question of functional complementation between mycorrhizal fungi and bacteria in the ectomycorrhizal complex for the promotion of tree nutrition.

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Petnicki-Ocwieja, Tanja, Tomas Hrncir, Yuen-Joyce Liu, Amlan Biswas, Tomas Hudcovic, Helena Tlaskalova-Hogenova, and Koichi S. Kobayashi. "Nod2 is required for the regulation of commensal microbiota in the intestine." Proceedings of the National Academy of Sciences 106, no. 37 (September 1, 2009): 15813–18. http://dx.doi.org/10.1073/pnas.0907722106.

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Mutations in the Nod2 gene are among the strongest genetic risk factors in the pathogenesis of ileal Crohn's disease, but the exact contributions of Nod2 to intestinal mucosal homeostasis are not understood. Here we show that Nod2 plays an essential role in controlling commensal bacterial flora in the intestine. Analysis of intestinal bacteria from the terminal ilea of Nod2-deficient mice showed that they harbor an increased load of commensal resident bacteria. Furthermore, Nod2-deficient mice had a diminished ability to prevent intestinal colonization of pathogenic bacteria. In vitro, intestinal crypts isolated from terminal ilea of Nod2-deficient mice were unable to kill bacteria effectively, suggesting an important role of Nod2 signaling in crypt function. Interestingly, the expression of Nod2 is dependent on the presence of commensal bacteria, because mice re-derived into germ-free conditions expressed significantly less Nod2 in their terminal ilea, and complementation of commensal bacteria into germ-free mice induced Nod2 expression. Therefore, Nod2 and intestinal commensal bacterial flora maintain a balance by regulating each other through a feedback mechanism. Dysfunction of Nod2 results in a break-down of this homeostasis.

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Perez-Castineira, J. R., R. L. Lopez-Marques, J. M. Villalba, M. Losada, and A. Serrano. "Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases." Proceedings of the National Academy of Sciences 99, no. 25 (November 25, 2002): 15914–19. http://dx.doi.org/10.1073/pnas.242625399.

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Xiang, Longkuan, and Bradley S. Moore. "Inactivation, Complementation, and Heterologous Expression ofencP, a Novel Bacterial Phenylalanine Ammonia-Lyase Gene." Journal of Biological Chemistry 277, no. 36 (June 24, 2002): 32505–9. http://dx.doi.org/10.1074/jbc.m204171200.

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Jahr, Holger, Jens Dreier, Dietmar Meletzus, Rainer Bahro, and Rudolf Eichenlaub. "The Endo-β-1,4-glucanase CelA of Clavibacter michiganensis subsp. michiganensis Is a Pathogenicity Determinant Required for Induction of Bacterial Wilt of Tomato." Molecular Plant-Microbe Interactions® 13, no. 7 (July 2000): 703–14. http://dx.doi.org/10.1094/mpmi.2000.13.7.703.

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The phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt and canker of tomato, harbors two plasmids, pCM1 (27.35 kb) and pCM2 (72 kb), encoding genes involved in virulence (D. Meletzus, A. Bermpohl, J. Dreier, and R. Eichenlaub, 1993, J. Bacteriol. 175:2131–2136; J. Dreier, D. Meletzus, and R. Eichenlaub, 1997, Mol. Plant-Microbe Interact. 10:195–206). The region of pCM1 carrying the endoglucanase gene celA was mapped by deletion analysis and complementation. RNA hybridization identified a 2.4-knt (kilonucleotide) transcript of the celA structural gene and the transcriptional initiation site was mapped. The celA gene encodes CelA, a protein of 78 kDa (746 amino acids) with similarity to endo-β-1,4-glucanases of family A1 cellulases. CelA has a three-domain structure with a catalytic domain, a type IIa-like cellulose-binding domain, and a C-terminal domain. We present evidence that CelA plays a major role in pathogenicity, since wilt induction capability is obtained by endoglucanase expression in plasmid-free, nonvirulent strains and by complementation of the CelA- gene-replacement mutant CMM-H4 with the wild-type celA gene.

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Verch, Thorsten, Zhen-Kun Pan, and Yvonne Paterson. "Listeria monocytogenes-Based Antibiotic Resistance Gene-Free Antigen Delivery System Applicable to Other Bacterial Vectors and DNA Vaccines." Infection and Immunity 72, no. 11 (November 2004): 6418–25. http://dx.doi.org/10.1128/iai.72.11.6418-6425.2004.

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ABSTRACT Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed a Listeria monocytogenes-Escherichia coli shuttle plasmid that is retained by complementation of d-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied the d-alanine racemase complementation system to our Listeria cancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the new Listeria vector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, the Listeria vaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.

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Vogne, Christelle, Julio Ramos Aires, Christiane Bailly, Didier Hocquet, and Patrick Plésiat. "Role of the Multidrug Efflux System MexXY in the Emergence of Moderate Resistance to Aminoglycosides among Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis." Antimicrobial Agents and Chemotherapy 48, no. 5 (May 2004): 1676–80. http://dx.doi.org/10.1128/aac.48.5.1676-1680.2004.

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ABSTRACT This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa. Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs. As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria. This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin. Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain. Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients. In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs. These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P. aeruginosa.

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King, Amy M., Gabriela Pretre, Thanatchaporn Bartpho, Rasana W. Sermswan, Claudia Toma, Toshihiko Suzuki, Azad Eshghi, Mathieu Picardeau, Ben Adler, and Gerald L. Murray. "High-Temperature Protein G Is an Essential Virulence Factor of Leptospira interrogans." Infection and Immunity 82, no. 3 (December 23, 2013): 1123–31. http://dx.doi.org/10.1128/iai.01546-13.

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ABSTRACTLeptospira interrogansis a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide. There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would aid in vaccine development and the prevention of infection. HtpG (high-temperatureproteinG) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of anL. interroganshtpGtransposon insertion mutant, this study demonstrates thatL. interrogansHtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of thehtpGmutant completely restored virulence. Surprisingly, thehtpGmutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common inhtpGmutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.

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Rodríguez-Andrade, Osvaldo, Andrés Corral-Lugo, Yolanda E. Morales-García, Verónica Quintero-Hernández, América P. Rivera-Urbalejo, Dalia Molina-Romero, Rebeca D. Martínez-Contreras, Patricia Bernal, and Jesús Muñoz-Rojas. "Identification of Klebsiella Variicola T29A Genes Involved In Tolerance To Desiccation." Open Microbiology Journal 13, no. 1 (September 30, 2019): 256–67. http://dx.doi.org/10.2174/1874285801913010256.

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Introduction: Several plant-beneficial bacteria have the capability to promote the growth of plants through different mechanisms. The survival of such bacteria could be affected by environmental abiotic factors compromising their capabilities of phytostimulation. One of the limiting abiotic factors is low water availability. Materials and Methods: In extreme cases, bacterial cells can suffer desiccation, which triggers harmful effects on cells. Bacteria tolerant to desiccation have developed different strategies to cope with these conditions; however, the genes involved in these processes have not been sufficiently explored. Klebsiella variicola T29A is a beneficial bacterial strain that promotes the growth of corn plants and is highly tolerant to desiccation. In the present work, we investigated genes involved in desiccation tolerance. Results & Discussion: As a result, a library of 8974 mutants of this bacterial strain was generated by random mutagenesis with mini-Tn5 transposon, and mutants that lost the capability to tolerate desiccation were selected. We found 14 sensitive mutants; those with the lowest bacterial survival rate contained mini-Tn5 transposon inserted into genes encoding a protein domain related to BetR, putative secretion ATPase and dihydroorotase. The mutant in the betR gene had the lowest survival; therefore, the mutagenized gene was validated using specific amplification and sequencing. Conclusion: Trans complementation with the wild-type gene improved the survival of the mutant under desiccation conditions, showing that this gene is a determinant for the survival of K. variicola T29A under desiccation conditions.

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Gangaiah, Dharanesh, Wei Li, Kate R. Fortney, Diane M. Janowicz, Sheila Ellinger, Beth Zwickl, Barry P. Katz, and Stanley M. Spinola. "Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms." Infection and Immunity 81, no. 2 (December 10, 2012): 608–17. http://dx.doi.org/10.1128/iai.01239-12.

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ABSTRACTThe carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence.Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog ofcsrA. Here, we generated an unmarked, in-frame deletion mutant ofcsrAto assess its contribution toH. ducreyipathogenesis. In human inoculation experiments, thecsrAmutant was partially attenuated for pustule formation compared to its parent. Deletion ofcsrAresulted in decreased adherence ofH. ducreyito human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants ofH. ducreyiadherence to HFF cells, were downregulated in thecsrAmutant. Compared to its parent, thecsrAmutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. ThecsrAmutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation intranspartially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

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Pollock, Thomas J., Wilbert A. T. van Workum, Linda Thorne, Marcia J. Mikolajczak, Motohide Yamazaki, Jan W. Kijne, and Richard W. Armentrout. "Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in Sphingomonas Strain S88 andRhizobium leguminosarum." Journal of Bacteriology 180, no. 3 (February 1, 1998): 586–93. http://dx.doi.org/10.1128/jb.180.3.586-593.1998.

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ABSTRACT Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene ofSphingomonas strain S88 and the pssDE genes ofRhizobium leguminosarum were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(β1→4)-glucuronosyl transferase inSphingomonas and that pssC codes for a glucuronosyl-(β1→4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

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Hani, Eric Kurt, David Ng, and Voon-Loong Chan. "Arginine biosynthesis inCampylobacter jejuniTGH9011: determination of theargCOBDcluster." Canadian Journal of Microbiology 45, no. 11 (November 1, 1999): 959–69. http://dx.doi.org/10.1139/w99-095.

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Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. GroupV is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.Key words: arginine synthesis, Campylobacter jejuni, arginine biosynthetic genes, gene sequence, gene arrangement.

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Angus, Annette A., David J. Evans, Joseph T. Barbieri, and Suzanne M. J. Fleiszig. "The ADP-Ribosylation Domain of Pseudomonas aeruginosa ExoS Is Required for Membrane Bleb Niche Formation and Bacterial Survival within Epithelial Cells." Infection and Immunity 78, no. 11 (August 23, 2010): 4500–4510. http://dx.doi.org/10.1128/iai.00417-10.

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ABSTRACT Pseudomonas aeruginosa can establish a niche within the plasma membrane of epithelial cells (bleb niches) within which bacteria can survive, replicate, and swim at speeds detectable by real-time phase-contrast imaging. This novel virulence strategy is dependent on the bacterial type three secretion system (T3SS), since mutants lacking the T3SS needle or known T3SS effectors localize to perinuclear vacuoles and fail to replicate. Here, we determined which of the three effectors (ExoS, ExoT, or ExoY) were required for bleb niche formation and intracellular replication. PAO1 strains with mutations in exoS, exoT, exoY, or combinations thereof were compared to wild-type and complemented strains. P. aeruginosa exoS mutants, but not exoT or exoY mutants, lost the capacity for bleb niche formation and intracellular replication. Complementation with exoS rescued both phenotypes, either in the background of an exoS mutant or in a mutant lacking all three known effectors. Complementation with activity domain mutants of exoS revealed that the ADP-ribosyltransferase (ADP-r) activity of ExoS, but not the Rho-GAP activity nor the membrane localization domain (MLD) of ExoS, was required to elicit this phenotype. Membrane bleb niches that contained P. aeruginosa also bound annexin V-enhanced green fluorescent protein (EGFP), a marker of early apoptosis. These data show that P. aeruginosa bleb niches and intracellular survival involve ExoS ADP-r activity and implicate a connection between bleb niche formation and the known role(s) of ExoS-mediated apoptosis and/or Rab GTPase inactivation.

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Fath, M. J., R. C. Skvirsky, and R. Kolter. "Functional complementation between bacterial MDR-like export systems: colicin V, alpha-hemolysin, and Erwinia protease." Journal of Bacteriology 173, no. 23 (1991): 7549–56. http://dx.doi.org/10.1128/jb.173.23.7549-7556.1991.

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Schick Zapanta, Laura, Takefumi Hattori, Magarita Rzetskaya, and Ming Tien. "Cloning of Phanerochaete chrysosporium leu2 by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs." Applied and Environmental Microbiology 64, no. 7 (July 1, 1998): 2624–29. http://dx.doi.org/10.1128/aem.64.7.2624-2629.1998.

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ABSTRACT A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in bothEscherichia coli and Saccharomyces cerevisiae. This expression vector, λYES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from P. chrysosporiumwas characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.

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Putnoky, Péter, Veronika Deák, Krisztina Békási, Adrienn Pálvölgyi, Anita Maász, Zsuzsanna Palágyi, Gyula Hoffmann, and Ildikó Kerepesi. "H Protein of Bacteriophage 16-3 and RkpM Protein of Sinorhizobium meliloti 41 Are Involved in Phage Adsorption." Journal of Bacteriology 186, no. 6 (March 15, 2004): 1591–97. http://dx.doi.org/10.1128/jb.186.6.1591-1597.2004.

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ABSTRACT The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.

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Asselin, Jo Ann E., Jean M. Bonasera, and Steven V. Beer. "Center Rot of Onion (Allium cepa) Caused by Pantoea ananatis Requires pepM, a Predicted Phosphonate-Related Gene." Molecular Plant-Microbe Interactions® 31, no. 12 (December 2018): 1291–300. http://dx.doi.org/10.1094/mpmi-04-18-0077-r.

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Pantoea ananatis, a cause of center rot of onion, is problematic in the United States and elsewhere. The bacterium lacks disease determinants common to most other bacterial pathogens of plants. A genomic island containing the gene pepM was detected within many onion-pathogenic strains of P. ananatis of diverse origins. The pepM gene of P. ananatis putatively encodes a protein that converts phosphoenolpyruvate to phosphonopyruvate, the first step in the biosynthesis of phosphonates and related molecules. This gene appears to be essential for center rot disease. Deletion of pepM rendered the mutant strain unable to cause lesions in leaves of growing onions and water-soaking of inoculated yellow onion bulbs. Furthermore, growth of the deletion mutant in onion leaves was significantly diminished compared with wild-type bacteria, and the mutant failed to cause cell death in tobacco. Complementation of the mutated strain with pepM restored the phenotype to wild-type capability. The pepM gene is the first pathogenicity factor identified that affects bacterial fitness as well as symptom development in both leaves and bulbs in a pathogen causing center rot of onion.

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Alqahtani, Fulwah, Jafar Mahdavi, Lee M. Wheldon, Matthew Vassey, Necmettin Pirinccioglu, Pierre-Joseph Royer, Suzan M. Qarani, et al. "Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis." Open Biology 4, no. 10 (October 2014): 140053. http://dx.doi.org/10.1098/rsob.140053.

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The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C 173 ) of Gal-3 or lysine (K 166 ) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria ( Neisseria meningitidis ), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

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Burbank, Lindsey P., and Drake C. Stenger. "Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection." Phytopathology® 106, no. 8 (August 2016): 928–36. http://dx.doi.org/10.1094/phyto-02-16-0097-r.

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The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

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Monteiro, Freddy, Montserrat Solé, Irene van Dijk, and Marc Valls. "A Chromosomal Insertion Toolbox for Promoter Probing, Mutant Complementation, and Pathogenicity Studies in Ralstonia solanacearum." Molecular Plant-Microbe Interactions® 25, no. 4 (April 2012): 557–68. http://dx.doi.org/10.1094/mpmi-07-11-0201.

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We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors—the Ralstonia chromosome (pRC) series—that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).

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Ianiri, Giuseppe, Marco A. Coelho, Fiorella Ruchti, Florian Sparber, Timothy J. McMahon, Ci Fu, Madison Bolejack, et al. "HGT in the human and skin commensalMalassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction." Proceedings of the National Academy of Sciences 117, no. 27 (June 23, 2020): 15884–94. http://dx.doi.org/10.1073/pnas.2003473117.

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The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions.Malasseziaare the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn’s disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within theMalasseziagenus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation inMalassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure ofMalasseziaflavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent ofMalasseziaflavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

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Washburn, Robert S., Andrea Marra, Alexander P. Bryant, Martin Rosenberg, and Daniel R. Gentry. "rho Is Not Essential for Viability or Virulence inStaphylococcus aureus." Antimicrobial Agents and Chemotherapy 45, no. 4 (April 1, 2001): 1099–103. http://dx.doi.org/10.1128/aac.45.4.1099-1103.2001.

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ABSTRACT We have identified the gene for transcription termination factor Rho in Staphylococcus aureus. Deletion ofrho in S. aureus reveals that it is not essential for viability or virulence. We also searched the available bacterial genomic sequences for homologs of Rho and found that it is broadly distributed and highly conserved. Exceptions includeStreptococcus pneumoniae, Streptococcus pyogenes, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum, and Synechocystis sp. strain PCC6803, all of which appear not to possess a Rho homolog. Complementation studies indicate that S. aureus Rho possesses the same activity as Escherichia coli Rho and that the Rho inhibitor bicyclomycin is active against S. aureus Rho. Our results explain the lack of activity of bicyclomycin against many gram-positive bacteria and raise the possibility that the essentiality of rho may be the exception rather than the rule.

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Page, Frederic, Silvia Altabe, Nicole Hugouvieux-Cotte-Pattat, Jean-Marie Lacroix, Janine Robert-Baudouy, and Jean-Pierre Bohin. "Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity." Journal of Bacteriology 183, no. 10 (May 15, 2001): 3134–41. http://dx.doi.org/10.1128/jb.183.10.3134-3141.2001.

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ABSTRACT Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGHoperon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgHgene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.

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Coulombe, François, Maziar Divangahi, Frédéric Veyrier, Louis de Léséleuc, James L. Gleason, Yibin Yang, Michelle A. Kelliher, et al. "Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide." Journal of Experimental Medicine 206, no. 8 (July 6, 2009): 1709–16. http://dx.doi.org/10.1084/jem.20081779.

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Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) α secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor κB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

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Chan, Wai, Mirian Domenech, Inmaculada Moreno-Córdoba, Verónica Navarro-Martínez, Concha Nieto, Miriam Moscoso, Ernesto García, and Manuel Espinosa. "The Streptococcus pneumoniae yefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation." Toxins 10, no. 9 (September 18, 2018): 378. http://dx.doi.org/10.3390/toxins10090378.

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Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein–protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

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Wang, Chunxia, David J. Meek, Priya Panchal, Natalie Boruvka, Frederick S. Archibald, Brian T. Driscoll, and Trevor C. Charles. "Isolation of Poly-3-Hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants." Applied and Environmental Microbiology 72, no. 1 (January 2006): 384–91. http://dx.doi.org/10.1128/aem.72.1.384-391.2006.

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ABSTRACT The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate d-3-hydroxybutyrate due to absence of the enzyme d-3-hydroxybutyrate dehydrogenase activity. Clones that conferred d-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored d-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.

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Rainczuk, Arek K., Stephan Klatt, Yoshiki Yamaryo-Botté, Rajini Brammananth, Malcolm J. McConville, Ross L. Coppel, and Paul K. Crellin. "MtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates." Journal of Biological Chemistry 295, no. 18 (March 26, 2020): 6108–19. http://dx.doi.org/10.1074/jbc.ra119.011688.

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Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

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Choi, Eunsil, Nalae Kang, Young Jeon, Hyun-Sook Pai, Sung-Gun Kim, and Jihwan Hwang. "Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation." Journal of Bacteriology 198, no. 17 (June 13, 2016): 2284–96. http://dx.doi.org/10.1128/jb.00384-16.

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ABSTRACTThe uniqueEscherichia coliGTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria,Klebsiella pneumoniaeandSalmonella entericaserovar Typhimurium; two pathogenic bacteria,Staphylococcus aureusandNeisseria gonorrhoeae; and the extremophileDeinococcus radioduransand then evaluated whether they could functionally complement theE. colider-null phenotype. OnlyK. pneumoniaeandS. Typhimurium Der proteins enabled theE. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression ofK. pneumoniaeandS. Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused byE. coliDer depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of theder-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit.IMPORTANCEInEscherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them,Klebsiella pneumoniaeandSalmonella entericaserovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly inE. coli. Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation inE. coli dermutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits.

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Rachid, Shwan, Knut Ohlsen, Ursula Wallner, Jörg Hacker, Michael Hecker, and Wilma Ziebuhr. "Alternative Transcription Factor ςB Is Involved in Regulation of Biofilm Expression in a Staphylococcus aureusMucosal Isolate." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6824–26. http://dx.doi.org/10.1128/jb.182.23.6824-6826.2000.

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ABSTRACT Osmotic stress was found to induce biofilm formation in aStaphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor ςB, resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding ςB completely restored the wild-type phenotype. The combined data suggest a critical role of ςB in S. aureus biofilm regulation under environmental stress conditions.

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Smiley, J. A., and D. K. Asch. "Identification of a gene encoding GMP synthetase from a Neurospora crassa cDNA library by bacterial complementation." Fungal Genetics Reports 47, no. 1 (July 25, 2000): 94–95. http://dx.doi.org/10.4148/1941-4765.1214.

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INOUYE, Takayuki, Masaki ODAHARA, Tomomichi FUJITA, Mitsuyasu HASEBE, and Yasuhiko SEKINE. "Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the MossPhyscomitrella patens." Bioscience, Biotechnology, and Biochemistry 72, no. 5 (May 23, 2008): 1340–47. http://dx.doi.org/10.1271/bbb.80014.

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Stevenson, Brian, and Kelly Babb. "LuxS-Mediated Quorum Sensing in Borrelia burgdorferi, the Lyme Disease Spirochete." Infection and Immunity 70, no. 8 (August 2002): 4099–105. http://dx.doi.org/10.1128/iai.70.8.4099-4105.2002.

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ABSTRACT The establishment of Borrelia burgdorferi infection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues. This complex process requires regulated synthesis of many bacterial proteins. We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete. The luxS gene of B. burgdorferi was identified and demonstrated to encode a functional enzyme by complementation of an Escherichia coli luxS mutant. Cultured B. burgdorferi responded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins. Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes.

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Journal articles on the topic 'Bacterial complementation'

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