Inhibition of Escherichia coli precursor‐16S rRNA processing by mouse intestinal contents

Abstract

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ. In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coil cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coil growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.

Mouse Intestine Selects Nonmotile flhDC Mutants of Escherichia coli MG1655 with Increased Colonizing Ability and Better Utilization of Carbon Sources

“…E. coli strains grow extremely well (generation times of 25 to 35 min, viable counts of about 10 9 CFU/ml) in cecal mucus (23,30,34,43,44,46). To determine whether the MG1655* and MG1655 ⌬edd* strains had a growth advantage in cecal mucus, MG1655, MG1655*, MG1655 ⌬edd, and MG1655 ⌬edd* were each inoculated separately into cecal mucus that had been diluted 50-fold into HEPES-Hanks buffer, pH 7.4 (1 mg/ml with respect to protein), at an A 600 of 0.1.…”

“…Since E. coli colonization of the mouse intestine appears to require the ability to grow in mucus (23,30,34,43,44,46), the power of DNA microarrays was used to focus attention on identifying genes induced by growth in mouse cecal mucus in vitro relative to growth in minimal medium containing glucose as the carbon source. This approach allowed the identification of additional nutrients, including N-acetyl-D-glucosamine, Nacetylneuraminic (sialic) acid, L-fucose, D-ribose, and D-glucuronate, as being necessary for the maximum ability of E. coli MG1655 to colonize the intestine (8).…”

Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses

“…rRNA concentration may correlate well with growth rate in some strains of bacteria, but correlations can differ significantly between strains (Wade and Robinson, 1965;Kemp et al, 1993;Pang and Winkler, 1994;Binnerup et al, 2001;Worden and Binder, 2003). Even at the 'species' level of bacteria, the relationship between rRNA and growth rate can differ significantly between subpopulations (Rosset et al, 1966;Licht et al, 1999). Hence, using rRNA to compare relative activity or changes in activity between taxa will likely provide misleading information.…”

“…For example, the relationship between microbial activity and measurable rRNA can be influenced by heterogeneity of cell physiology within a population (Licht et al, 1999), changes in the ratio of non-growth to growth-specific metabolic activity, life history (Oda et al, 2000), life strategy (Flärdh et al, 1992;Lepp and Schmidt, 1998;Mitchell et al, 2009;Sukenik et al, 2012), sample heterogeneity, changing environmental conditions and of course fundamental enzyme kinetics (that is, substrate concentration). Additionally, the concentration of rRNA in a cell at a given point in time is the net result of rRNA synthesis (that is, transcription) and degradation rates (Gausing, 1977), each of which may be under distinct controls.…”

Application of molecular tools for in situ monitoring of bacterial growth activity

“…The fast growth estimated by the ribosome counting method was totally incompatible with the size of the intestinal E. coli population and the faecal excretion rate. This paradox was eventually resolved in a further study of the compartmentalization of the bacteria in the mouse gut (Licht et al, 1999), which demonstrated the presence of two different subpopulations of E. coli : one small population residing in the mucus, which was growing fast, and another much larger non-growing population located in the intestinal lumen, which seemed to be inhibited by an unknown microbial antagonist interfering with the growth of the E. coli cells. The two E. coli subpopulations had similar ribosomal contents.…”