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Stratified Growth in Pseudomonas aeruginosa Biofilms. Erin Werner, 2004.In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms . Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs . One construct carried an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP . The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp1 promoter . Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen . The zone of active GFP expression was approximately 60 µm wide in colony biofilms and 30 µm wide in flow cell biofilms . The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy . Cell elongation was localized at the air interface of the biofilm . The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm . Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 µm into the biofilm . P . aeruginosa was incapable of anaerobic growth in the medium used for this investigation . These results show that while mature P . aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing . Tet(L) and Tet(K) Tetracycline-Divalent Metal/H+ Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences. Jie Jin, 2002.The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me2+) complex that bears a net single positive charge, Na+, and K+ . Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion . In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated . Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux . Here they were shown to couple TC-Me2+ efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased . The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion . Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative "antiporter motif," motif C, of Tet proteins would be involved in these characteristic preferences . Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate . A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference . Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion . However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates . Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis. Irina Bagyan, 2002.The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis . CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins . However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca2+-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant . These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins .
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