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Insertional Inactivation (insertional + inactivation)
Selected Abstractsd -Alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100-23 results in impaired colonization of the mouse gastrointestinal tractENVIRONMENTAL MICROBIOLOGY, Issue 7 2007Jens Walter Summary The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of d -alanine esters into cell wall-associated teichoic acids (TA). d -Alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of d -alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of d -alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex- Lactobacillus -free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that d -alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach. [source] Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridiaMOLECULAR MICROBIOLOGY, Issue 5 2000Adriana Ravagnani The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1.4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth. [source] Listeria monocytogenes response regulators important for stress tolerance and pathogenesisFEMS MICROBIOLOGY LETTERS, Issue 1 2001Birgitte H. Kallipolitis Abstract Environmental sensing by two-component signal transduction systems is likely to play a role for growth and survival of Listeria monocytogenes both during transmission in food products and within a host organism. Two-component systems typically consist of a membrane-associated sensor histidine kinase and a gene regulatory protein, the response regulator (RR). We have identified seven putative RR genes in L. monocytogenes LO28 by PCR using degenerate oligonucleotide primers. By insertional inactivation we obtained data suggesting that three of the putative RRs contribute to the pathogenicity of L. monocytogenes in mice. Strikingly, the mutants that were attenuated in virulence also had a decreased ability to grow in the presence of various stress conditions potentially encountered in an infection process. Thus, our data point to a connection between the ability of the putative two-component systems to sense and respond to certain environmental stimuli, and the virulence of L. monocytogenes. [source] Effects of recombinase A deficiency on biofilm formation by Streptococcus mutansMOLECULAR ORAL MICROBIOLOGY, Issue 2 2009S. Inagaki Background/aim:, Recombinase A (RecA) is essential for the transformation of both plasmid and chromosomal DNA in Streptococcus pneumoniae and is considered to be related to the SOS-response in Streptococcus mutans. Methods:, In the present study, a RecA-deficient mutant strain (RAD) was constructed by insertional inactivation of the recA gene encoding the RecA protein in strain MT8148 of S. mutans, after which the biological functions of acid tolerance and biofilm formation were investigated. Results:, RAD showed reduced acid tolerance and produced lower density biofilm compared with the wild-type strain. In addition, confocal microscopic observation indicated that the biofilm produced by RAD was composed of cells with significantly lower viability compared with that produced by strain MT8148. Conclusion:, These results suggest that RecA has a relationship with biofilm formation. [source] WRKY70 modulates the selection of signaling pathways in plant defenseTHE PLANT JOURNAL, Issue 3 2006Jing Li Summary Cross-talk between signal transduction pathways is a central feature of the tightly regulated plant defense signaling network. The potential synergism or antagonism between defense pathways is determined by recognition of the type of pathogen or pathogen-derived elicitor. Our studies have identified WRKY70 as a node of convergence for integrating salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling events during plant response to bacterial pathogens. Here, we challenged transgenic plants altered in WRKY70 expression as well as WRKY70 knockout mutants of Arabidopsis with the fungal pathogens Alternaria brassicicola and Erysiphe cichoracearum to elucidate the role of WRKY70 in modulating the balance between distinct defense responses. Gain or loss of WRKY70 function causes opposite effects on JA-mediated resistance to A. brassicicola and the SA-mediated resistance to E. cichoracearum. While the up-regulation of WRKY70 caused enhanced resistance to E. cichoracearum, it compromised plant resistance to A. brassicicola. Conversely, down-regulation or insertional inactivation of WRKY70 impaired plant resistance to E. cichoracearum. Over-expression of WRKY70 resulted in the suppression of several JA responses including expression of a subset of JA- and A. brassicicola -responsive genes. We show that this WRKY70 -controlled suppression of JA-signaling is partly executed by NPR1. The results indicate that WRKY70 has a pivotal role in determining the balance between SA-dependent and JA-dependent defense pathways. [source] Evidence for efflux pumps, other than PmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniaeCLINICAL MICROBIOLOGY AND INFECTION, Issue 2 2003N. P. Brenwald Fluoroquinolone resistance in pneumococci is known to be associated with the efflux pump, PmrA. However, there may be other efflux systems that also cause drug resistance. Two types of mutants were studied. The efflux phenotype from mutants selected by sub-MIC levofloxacin or gemifloxacin was transformed into R6. These transformants did not show increased pmrA transcripts in Northern blots; insertional inactivation of pmrA in the transformants did not abolish the efflux phenotype. A second set of efflux phenotype mutants was selected in R6:cat by ethidium bromide but not by norfloxacin; accumulation of ethidium bromide in the one among these mutants studied was reduced in comparison to its parent. This evidence suggests that systems other than PmrA can contribute to efflux-mediated resistance in pneumococci. [source] | |||||||||||||