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Sinorhizobium meliloti ExoR and ExoS Proteins Regulate both Succinoglycan and Flagellum Production. Shi-Yi Yao, 2004.The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa [Medicago sativa] by S . meliloti . Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproductionof succinoglycan and a reduction in symbiosis . Systematic analysesof the symbiotic phenotypes of the two mutants demonstratedtheir reduced efficiency of root hair colonization . In addition,both the exoR95 and exoS96 mutations caused a marked reductionin the biosynthesis of flagella and consequent loss of abilityof the cells to swarm and swim . Succinoglycan overproductiondid not appear to be the cause of the suppression of flagellumbiosynthesis . Further analysis indicated that both the exoR95and exoS96 mutations affected the expression of the flagellumbiosynthesis genes . These findings suggest that both the ExoRprotein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis . These findings provide new avenues of understandingof the physiological changes S . meliloti cells go through during the early stages of symbiosis and of the signal transductionpathways that mediate such changes. Effect of Glycine on Helicobacter pylori In Vitro. Masaaki Minami, 2004.Glycine is the simplest amino acid and is used as a metabolic product in some bacteria . However, an excess of glycine inhibits the growth of many bacteria, and it is used as a nonspecific antiseptic agent due to its low level of toxicity in animals . The effect of glycine on Helicobacter pylori is not precisely known . The present study was conducted to investigate (i) the effect of glycine on clarithromycin (CLR)-resistant and -susceptible strains of H . pylori, (ii) the effect of glycine in combination with amoxicillin (AMX), and (iii) the postantibiotic effect (PAE) . The MIC at which 90% of strains are inhibited for glycine was almost 2.5 mg/ml for 31 strains of H . pylori, including CLR-resistant strains . We constructed isogenic CLR-resistant mutant strains by natural transformation and investigated the difference between clinical wild-type strains and isogenic mutants . There were no differences in the MICs between CLR-resistant and -susceptible strains or between clinical wild-type and mutant strains . The combination of AMX and glycine showed synergistic activity, with the minimum bactericidal concentration of AMX with glycine decreasing to 1/10 that of AMX alone . Glycine showed no PAE against H . pylori . These results suggest that glycine may be a useful antimicrobial agent against H . pylori not only alone but also in combination with antibacterial drugs for the treatment of H . pylori-associated diseases . Glycine may represent a component of a new type of eradication therapy for CLR-resistant H . pylori . Antianaerobic Activity of a Novel Fluoroquinolone, WCK 771, Compared to Those of Nine Other Agents. Mihaela Peric, 2004.Agar dilution MIC methodology was used to compare the activity of WCK 771 with those of ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, piperacillin, piperacillin-tazobactam, imipenem, clindamycin, and metronidazole against 350 anaerobes . Overall, the MICs (in micrograms per milliliter) at which 50 and 90%, respectively, of the isolates tested were inhibited were as follows: WCK 771, 0.5 and 2.0; ciprofloxacin, 2.0 and 32.0; levofloxacin, 1.0 and 8.0; gatifloxacin, 0.5 and 4.0; moxifloxacin, 0.5 and 4.0; piperacillin, 2.0 and 64.0; piperacillin-tazobactam,  0.125 and 8.0; imipenem, 0.125 and 1.0; clindamycin, 0.125 and 16.0; and metronidazole, 1.0 and >16.0 .
Activation of Prophage eib Genes for Immunoglobulin-Binding Proteins by Genes from the IbrAB Genetic Island of Escherichia coli ECOR-9. Carol H. Sandt, 2002.Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E . coli reference (ECOR) strain ECOR-9 . Each eib gene was transferred to test E . coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage . The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack . Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens . The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E . coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933) . The genomic segment containing ibrA and ibrB has been designated the IbrAB island . It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes . The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E . coli K-12 genome . Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2 . Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by  0.1% from ECOR-9, whereas eib-negative ECOR-47 diverges by 16% .
The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis. Ikuo Kojima, 2002.The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes . Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic . A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB . Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration . A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (  ), thus leading to the conclusion that orf5 was rpoZ in S . kasugaensis . The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete
of 47 amino acids . Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB . When rpoZ of S . kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences . This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S . kasugaensis and responsible for pleiotropy .
Suppressor Mutations in the Study of Photosystem I Biogenesis: sll0088 Is a Previously Unidentified Gene Involved in Reaction Center Accumulation in Synechocystis sp . Strain PCC 6803. Jianping Yu, 2003.In previous work, some members of our group isolated mutant strains of Synechocystis sp . strain PCC 6803 in which point mutations had been inserted into the psaC gene to alter the cysteine residues to the FA and FB iron-sulfur clusters in the PsaC subunit of photosystem I (J . P . Yu, I . R . Vassiliev, Y . S . Jung, J . H . Golbeck, and L . McIntosh, J . Biol . Chem . 272:8032-8039, 1997) . These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll a and photosystem I . In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 µmol m-2 s-1) . Two separate suppressor strains of C14SPsaC, termed C14SPsaC-R62 and C14SPsaC-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the Synechocystis sp . strain PCC 6803 genome named sll0088 . C14SPsaC-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482  C change in sll0088, and C14SPsaC-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in sll0088 . These suppressor strains showed near-wild-type levels of chlorophyll a and photosystem I, yet the serine oxygen ligand to FB was retained as shown by the retention of the S ≥ 3/2 spin state of the [4Fe-4S] cluster . The inactivation of sll0088 by insertion of a kanamycin resistance cartridge in the primary C14SPsaC mutant produced an engineered suppressor strain capable of photoautotrophic growth . There was no difference in psaC gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an sll0088 deletion mutant . The sll0088 gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms, including Arabidopsis thaliana . The protein contains a typical helix-turn-helix DNA-binding motif and can be classified as a negative regulator by phylogenetic analysis . This suggests that the product of sll0088 has a role in regulating the biogenesis of photosystem I .
Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in Cryosections of Microarthropods. Torsten Thimm, 2003.A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy . The protocol is based on cryosections of whole specimens . In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material . The method allowed the study of the bacterial colonization of Folsomia candida (Collembola) and the detection of bacteria in both the gut and tissue . Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast. Claudia A. Martins, 2003.Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species . The autonomous production of PSTs by bacteria remains controversial . In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods . Analyses were performed under conditions determined previously to be optimal for toxin production and detection . Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains .
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