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Archives of Microbiology (v.172, #1)
Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion by P. Genevaux; Pascale Bauda; Michael S. DuBow; Bauke Oudega (pp. 1-8).
Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed.
Keywords: Key words Bacterial adhesion; Biofilm formation; Abiotic surface; Type 1 fimbriae; Flagella; Lipopolysaccharide
Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor by Galina V. Mukamolova; Svetlana S. Kormer; Douglas B. Kell; A. S. Kaprelyants (pp. 9-14).
Viable cells of Micrococcus luteus secrete a proteineous growth factor (Rpf) which promotes the resuscitation of dormant, nongrowing cells to yield normal, colony-forming bacteria. When washed M. luteus cells were used as an inoculum, there was a pronounced influence of Rpf on the true lag phase and cell growth on lactate minimal medium. In the absence of Rpf, there was no increase in colony-forming units for up to 10 days. When the inoculum contained less than 105 cells ml–1, macroscopically observable M. luteus growth was not obtained in succinate minimal medium unless Rpf was added. Incubation of M. luteus in the stationary phase for 100 h resulted in a failure of the cells to grow in lactate minimal medium from inocula of small size although the viability of these cells was close to 100% as estimated using agar plates made from lactate minimal medium or rich medium. The underestimation of viable cells by the most-probable-number (MPN) method in comparsion with colony-forming units was equivalent to the requirement that at least 105 cells grown on succinate medium, 103 cells from old stationary phase, or approximately 10–500 washed cells are required per millilitre of inoculum for growth to lead to visible turbidity. The addition of Rpf in the MPN dilutions led to an increase of the viable cell numbers estimated to approximately the same levels as those determined by colony-forming units. Thus, a basic principle of microbiology –“one cell-one culture”– may not be applicable in some circumstances in which the metabolic activity of “starter” cells is not sufficient to produce enough autocrine growth factor to support cell multiplication.
Keywords: Key words Dormancy; Resuscitation; Latency; Anabiosis; Growth factor; Lag phase; Cell; multiplication; Micrococcus luteus; Mycobacterium; tuberculosis
Characterization of IS1389, a new member of the IS3 family of insertion sequences isolated from Xanthomonas campestris pv. amaranthicola by Pilar Gómez; R. M. Ribas-Aparicio; A. I. Peléez; M. Rosario Rodicio (pp. 15-21).
IS1389, a new insertion sequence belonging to the IS3 family, has been identified in Xanthomonas campestris pv. amaranthicola. The genome of this bacterium contains at least 11 copies of the element, whereas no hybridizing sequences were detected in other Xanthomonas species [X. axonopodis, X. fragaridae, X. phaseoli, and X. (Stenotrophomonas) maltophila]. Two nearly identical copies of the element (IS1389-A and IS1389-B) were characterized. According to analysis of sequence alignments and similar structural features, IS1389 belongs to the IS407 subgroup of the IS3 family, which duplicates 4 bp of target DNA upon insertion. IS1389-A was found in the proximity of the modification gene of the XamI restriction-modification system.
Keywords: Key words IS1389; Insertion sequence; IS3 family; XamI restriction-modification system; Xanthomonas campestris pv. amaranthicola
Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment by S. Niemann; Tanja Dammann-Kalinowski; Anja Nagel; Alfred Pühler; W. Selbitschka (pp. 22-30).
The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates.
Keywords: Key words Repetitive sequences; ERIC elements; Natural populations; Polymerase chain reaction; Rhizobium meliloti; Sinorhizobium medicae; Symbiotic nitrogen fixation
Functional assembly of the λ S holin requires periplasmic localization of its N-terminus by Anton Graschopf; U. Bläsi (pp. 31-39).
Bacteriophage-λ-induced host-cell lysis requires two phage-encoded proteins, the S holin and the R transglycosylase. At a specific time during infection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan. The λS gene represents the prototype of holin genes with a dual-start motif; they encode two proteins, a lysis effector and a lysis inhibitor. Although the two S proteins differ only by two amino acids (Met-1 and Lys-2) at the N-terminus, the longer product (S107) acts as an inhibitor of the lysis effector (S105). The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107. It was therefore of interest to determine the subcellular localization of the N-terminus of either S protein. To study the membrane topology of the S proteins, we used the topology probe TEM β-lactamase and an N-terminal tag derived from the Pseudomonas aeruginosa phage Pf3 coat protein. We show that both S proteins have a type III (Nout/Cin) topology. The results provide insight into the regulatory mechanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent “hole” in the membrane.
Pheophytinization of bacteriochlorophyll c and energy transfer in cells of Chlorobium tepidum by S. Tokita; Masamitsu Hirota; Niels-Ulrik Frigaard; Keizo Shimada; Katsumi Matsuura (pp. 40-44).
Bacteriochlorophyll (BChl) c in whole cells of Chlorobium tepidum grown at 46 °C changed into bacteriopheophytin (BPhe) c within 10 days after reaching full growth. When a small amount of C. tepidum cells in which BChl c had been completely pheophytinized were transferred to a new culture medium, normal growth was observed after a short lag phase, and the absorption spectrum of the growing cells showed the presence of a normal amount of BChl c. During the growth of C. tepidum in the new culture, the BChl c concentration was nearly proportional to the cell density measured by turbidity (OD640). These results indicate that C. tepidum can survive even when BChl c has been completely pheophytinized and that BChl c is newly synthesized in such cells when transferred to a new culture medium. In partly pheophytinized cells, upon excitation of BPhe c at 550 nm the fluorescence emission spectrum showed maxima at 775 and 810 nm, which correspond to emissions from BChl c and BChl a, respectively. This indicates energy transfer from BPhe c to BChl c and BChl a. In cells in which BChl c was completely pheophytinized, fluorescence measurements were indicative of direct energy transfer from BPhe c to baseplate BChl a. These findings suggest that when BChl c in C. tepidum cells is pheophytinized, the product (BPhe c) remains in the chlorosomes and continues to work as a light-harvesting pigment.
Keywords: Key words Bacteriochlorophyll c; Bacteriochlorophyll a; Bacteriopheophytin c; Chlorobium tepidum; Chlorosome; Energy transfer
Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions by Claudia Ludwig; Susanne Ecker; Kerstin Schwindel; Hans-Georg Rast; Karl Otto Stetter; Günther Eberz (pp. 45-50).
Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41–3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.
Keywords: Key words Ammonia oxidation; Bioluminescence; Conjugation; Nitrification inhibitor; Nitrosomonas
Characterization of the interaction of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum with bacterial membranes by Cale M. Halbleib; P. W. Ludden (pp. 51-58).
The interaction of dinitrogenase reductase-activating glycohydrolase (DRAG) with bacterial membranes and the solubilization of DRAG in response to nucleotides were characterized. Purified DRAG from Rhodospirillum rubrum reversibly bound bacterial pellet fractions from Rsp. rubrum and other nitrogen-fixing bacteria. DRAG saturated the membrane fraction of Rsp. rubrum at a concentration of 0.2 mol DRAG/mol bacteriochlorophyll, suggesting that the DRAG-binding species is prevalent in the membrane. DRAG bound poorly to phospholipid vesicles, suggesting a protein requirement for DRAG interaction with the membrane. Guanosine and uridine tri- and di-nucleotides specifically dissociated DRAG from the pellet fractions of Rsp. rubrum and Azotobacter vinelandii, while adenosine nucleotides had no dissociative effect. Guanosine 5′-triphosphate dissociated DRAG from the membrane at a concentration causing 50% dissociation (EC50) of 5.0 ± 0.5 mM; guanosine disphosphate had an EC50 of 15.0 ± 2.0 mM. We propose that GTP is a potential participant in the regulation of DRAG, possibly controlling the extent of DRAG association with the membrane.
Keywords: KeywordsRhodospirillum rubrum; Membrane; association; Nitrogenase regulation; Nucleotide binding
Discontinuous expansion linked to sector formation in Pseudomonas aeruginosa colonies by Annic Grondin; Harold C. Jarrell; L. R. Bérubé (pp. 59-62).
Colonies of Pseudomonas aeruginosa exhibit sectors that were shown to be located at specific intervals within the colony. Maxima in the distribution of sectors were observed every 5 mm as measured from the center of the colony. These maxima correlated with changes in the expansion rates of colonies. The absolute average number of sectors per colony was higher for colonies grown at higher temperatures. These results increase our understanding of colony pattern formation.
Keywords: Key wordsPseudomonas aeruginosa; Colony; development; Bacterial differentiation; Colony growth rates; Colony sectoring
Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds by Y. Mizunoe; S. N. Wai; Akemi Takade; Shin-ichi Yoshida (pp. 63-67).
Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 °C. Plate counts declined from 3 × 106 to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or α-ketoglutaric acid, plate counts increased to 104–105 CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H2O2, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells.
Keywords: Key wordsEscherichia coli O157; Nonculturable; cells; Restoration of culturability; H2O2-degrading; compounds