Although the DNA sequences of the seven P. Under all of the conditions tested, rrn E showed the highest level of activity. Nucleotide sequence alignment of the upstream sequences of seven UW4 rrn operons.
At different growth phases, three of the seven UW4 rrn promoters exhibited differential expression in E. The rrn A promoter showed a significantly higher expression level during mid-log phase than during early-log or stationary phases.
In the cases of rrn B, the highest expression occurred at early-log phase. The rrn C promoter showed significant differences between early- and mid-log phases. However, rrn D , rrn E , rrn F and rrn G displayed no significant differences among different growth phases. These results indicate that certain promoters are likely preferentially utilized at different temperatures.
When the promoters were tested in minimal medium in comparison with rich medium, only rrn A , rrn E and rrn F displayed significant differences. The rrn A promoter was downregulated, whereas rrn E and rrn F were upregulated in the minimal medium. This result is consistent with the possibility that some of these promoters play a more important role in protein synthesis under starvation conditions.
Some conclusions can be drawn from the differential expression and an analysis of the promoter sequences. For example, the promoter region of rrn F and rrn G showed It is possible that other elements might be involved in the regulation of rrn operons, including repressors or activators.
Nucleotide sequence alignment of the promoter region of rrn in Pseudomonas fluorescens F Accession no. Duan, K. Van Den Ham, B. Glick, J. Heikkila, unpublished data. Heat shock proteins in bacterial cells are induced when cells are exposed to environmental stresses such as elevated temperature, organic solvents, heavy metals, hydrogen peroxide, high pressure and extreme pH VanBogelen et al. The results presented in this study show that the promoters of P.
UW4 rrn are not functionally equivalent. Similar phenomenon was demonstrated in another Pseudomonas strain, P. In assays examining the effect of different development growth stages, temperatures and nutrition levels, it was found that different UW4 rrn promoters are preferentially activated.
This may confer an advantage on P. UW4 in the environment through fine-tuned control of protein expression in varying environmental situations and developmental stages. Appl Environ Microbiol 70 : — Google Scholar. Soil Biol Biochem 30 : — Can J Microbiol 53 : — Microbiol Mol Biol Rev 68 : — PLoS One 8 : e Plant Soil : — Environ Pollut : — Appl Environ Microbiol 63 : — Glick BR The enhancement of plant growth by free-living bacteria.
Can J Microbiol 41 : — Plant Physiol Biochem 39 : 11 — J Biotechnol 81 : 45 — Biochem Biophys Res Commun : — Hanahan D Studies on transformation of Escherichia coli with plasmids. J Mol Biol : — Curr Microbiol 41 : — Can J Plant Pathol 8 : — Google Preview. Plant Physiol Biochem 40 : — Appl Environ Microbiol 76 : — Can J Microbiol 44 : — Environ Microbiol 1 : — Solovyev V Salamov A Automatic annotation of microbial genomes and metagenomic sequences.
Plant Physiol Biochem 43 : — J Microbiol Methods 73 : — The number of ribosomes per cell is proportional to the growth rate to satisfy the cells demand for protein synthesis [ 3 ].
In fast growing E. The intracellular level of RNA polymerase, the key apparatus of transcription, correlates the ribosome level [ 5 , 6 ]. The syntheses of 3 rRNA species and all r-protein species are generally coordinated for efficient assembly of ribosomes. Each of the seven rRNA operons is transcribed from two promoters, upstream P1 and downstream P2, which are separated by approximately bp in length [ 9 ]. P1 plays a major role in high-level synthesis of rRNA during exponential growth in nutrient-rich media while P2 accounts for basal-level synthesis of rRNA at low growth rate and in stationary phase of cell growth.
Seven rRNA operons exist in more than 2, E. Strain W carries an inversion between rrnD and rrnE operons [A], but the direction of transcription for all seven rrn operons are the same with that of DNA replication in both strains.
Details are described in text. Even though the sequences of seven rrn operons are almost identical, significant difference exists in the promoter sequence between seven tRNA operons, and the rRNA sequences encoded by seven rrn operons include operon-specific minor difference see Discussion.
Seven rrn operons in E. The redundancy of rRNA operons in E. All seven rRNA operons are needed for optimal adaptation to changing physiological conditions [ 21 ]. After systematic approaches of making E. The correlation between the number of rrn operons and the rate of cell growth was also confirmed by using a set of engineered rRNA opeon copy-number variants [ 24 ].
These findings indicated that E. In fact, unused ribosomes are stored in inactive forms by forming ribosome dimers after interaction with dimerization factors such as ribosome modulation factor RMF [ 25 ].
The seven rRNA copies have long been believed to be identical in the structure and function. This concept was challenged after analysis of the expression mode of seven rRNA operons in E. Initial attempts were made to estimate the difference in the promoter activity of each rRNA operon using the protein reporter systems. Condon et al.
Since RNA genes lack the signal for translation, the reporter gene-associated SD sequence was used for expression of the reporter proteins in these studies.
Nevertheless, these pioneering studies suggested the growth rate- and growth phase-dependent difference in the expression pattern between seven rRNA operons.
These direct fusions between each rRNA promoter-GFP coding sequence, a kind of translational fusion, allowed more accurate estimation of the strength and regulation of rRNA promotors. Results herein described indicate that: the promoter activity is maximum for the rrnE operon; but the order of promoter strength between other six rrn operon promoters is different depending on the growth conditions. The molecular basis of the difference in promoter strength and regulation is discussed in relation of possible difference in the physiological role of each rrn operon.
The Glu tRNA gene is not present outside the rrn operons, implying that one evolutionary force for maintenance of the rrnB , rrnC , rrnD and rrnG operons is the inclusion of Glu-tRNA gene within these composite rrn operons. To confirm these proposals, we analyzed the number of Fis-binding sites along each bp-long rRNA promoter segment. Based on the gel shift assay, the number of Fis-binding sites on rrnA , rrnb , rrnC , rrnD and rrnH were estimated to be the same as reported [ 27 ], but that on rrnE increased from 5 to more than 8, and that on rrnG increased from 3 to more than 6 Fig 3.
Based on the gel shift pattern, the apparent increase in Fis-binding sites on these two promoters was suggested to be attributable to the cooperative binding of Fis at high protein concentrations. Open box and closed boxes on each probe represent the relative location of UP element and predicted Fis-binding sites, respectively see Fig 8. Triangles downstream of the UP element indicate two promoters, upstream P1 and downstream P2. The number of Fis sites on the rrnE promoter was suggested to be more than those hitherto identified see Fig 3.
The whole length used for the construction of pGRS vector is described in parenthesis at right-side end of each lane. Gel shift assay of Fis-binding sites was performed under the standard procedure as described in Materials and Methods using each of seven rrn promoter probes as shown in Fig 2 and increasing amounts of purified Fis protein.
With the rrnE and rrnG promoters, Fis exhibited high-level of cooperative binding, forming multiple ladders. Previously we constructed the quantitative promoter assay systems using GFP-RFP two-fluorescent protein reporters [ 28 ].
For determination of the promoter strength and regulation of seven rrn operons, approximately bp-long DNA fragments indicated by blue; for details see Fig 2 covering each of the rrn promoters, as shown in Fig 2 , were PCR-amplified and inserted into pGRS vector the modified version of pGRP vector with an inserted SD sequence indicated by yellow. Using the newly developed vectors for the assay of RNA promoters thus constructed, we first determined growth phase-dependent changes in the promoter activity of each rRNA operon.
Each of seven pGRS vectors was transformed into wild-type E. The level of promoter activity was the highest in the exponential growth phase at 2 hr after inoculation of the preculture to fresh media for all seven rRNA promoters and then decreased upon entry into stationary phase. The high-level activity of rrn promoters agrees with the high-level production of ribosomes in growing phase. The rate of decrease in promoter activity was relatively slow for pGRS-rrnC rrnC promoter and pGRS-rrnE rrnE promoter , and thus the GFP fluorescence still remained at detectable levels even at 20 and 30 hr after inoculation of the over-night preculture to a fresh medium.
Promoter activity of seven rrn operons was determined using a series of pGRS vector, two fluorescent reporter assay vectors for the plasmid map see Fig 4 , which were transformed into E. The growth curve dotted lines was determined by measuring turbidity the promoter activity straight lines was determined by measuring the fluorescent intensity and is shown as the relative values with the reference of lacUV5 -directed activity.
The level of rrn promoter activity was then compared for cells growing at different rates. The growth rates were: 1. For each culture, the promoter activity was measured as the relative value to that of reference lacUV5 promoter-directed activity purple line for each panel in Figs 5 — 7 at various times up to 30 hr after inoculation of the preculture to each fresh medium. Promoter activity of seven rrn operons was determined for the culture grown in LB red and M9-glucose orange media.
Growth curve and promoter activity were measured as in Fig 5 , and are shown in red LB and orange M9-Glc. Promoter activity of seven rrn operons was determined for the culture grown in LB red and M9-glycerol blue media. Growth curve and promoter activity were measured as in Fig 5 , and are shown in red LB and blue M9-Gly. In the growth phase-dependent change in the promoter activity for the M9-glucose-casamino acid culture is slightly different from that in LB culture Fig 5 , red line.
As in the case of LB culture, the maximum activity was observed at the 2 hr for all seven rRNA promoters Fig 5 , green line. The growth phase-coupled decrease in the activity of rrn promoters was observed for the slowly growing culture in M In both of these slowly growing cultures, the maximum promoter activity was observed at the culture time 5 hr.
The delay in the appearance of rrn promoter activity is mainly attributable to the decreased rate of cell growth in nutrient-poor media. The promoter activity was calculated at different times for all four cultures grown in different media.
The highest levels of promoter activity are compared between these different cultures Fig 8A. The difference in promoter strength ranged 6—7 fold between the strongest rrnE and the weakest rrnH. Travers R. S Martin P. W Reichchelderfer C. F Selective process for efficient isolation of soil Bacillus spp. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.
Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Alterra, P. Box , DK Roskilde, Denmark. Oxford Academic. Jaap Bloem. Bjarne Munk Hansen. Wim Wolters. Meint Veninga. Michael Hansen. Revision received:. Select Format Select format. Permissions Icon Permissions. Abstract The correlation between the growth rate, rRNA concentration and number of rrna operons was studied in Alcaligenes sp.
Open in new tab Download slide. Open in new tab. Growth rate regulation of rRNA content of a marine Synechococcus cyanobacterium strain. Google Scholar PubMed. Bacterial growth on surfaces: Automated image analysis for quantification of growth rate-related parameters.
Estimating the growth rate of slowly growing marine bacteria from RNA content. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy. Implications of rRNA operon copy number and ribosome content in the marine oligotrophic ultramicrobacterium Sphingomonas sp. Small ribosomal RNA content in marine proteobacteria during non-steady-state growth.
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Published by Elsevier Science B. All rights reserved. Issue Section:. Download all slides. View Metrics. Email alerts Article activity alert. Advance article alerts. New issue alert. In progress issue alert. Receive exclusive offers and updates from Oxford Academic. More on this topic Plant age and genotype affect the bacterial community composition in the tuber rhizosphere of field-grown sweet potato plants. Only the exponential phase data were used because that growth phase was the one examined by previous studies.
Ratios from MED and R. In contrast, ratios and growth rates for Ca. To explore differences between oligotrophic and copiotrophic bacteria in more detail, all of the data from copiotrophic bacteria were used in a linear regression analysis which was then used to examine the two oligotrophic bacteria.
The points for the oligotrophic bacteria are substantially below the regression line. Ratios for R. SAR92 was the least abundant of the groups examined in the estuary and was present in only 4 of 24 transect samples data not shown.
Ratios for Ca. The mean community growth rate in the estuary, calculated from bacterial production and abundance, was 0. There is a positive relationship between rRNA:rDNA and growth rate in many bacteria, but the bacteria with known relationships are not representative of dominant members of marine bacterial communities Kemp et al.
In particular, it is not clear whether the relationship between rRNA:rDNA and growth rate for oligotrophic bacteria, most notably the SAR11 clade, is similar to the relation known for copiotrophic bacteria. We determined the rRNA:rDNA ratio versus growth rate relationship in culture for four diverse marine bacteria that are representative of abundant clades in marine waters.
The rRNA:rDNA ratio is expected to depend on growth rate because increased ribosome content enables more protein synthesis and faster growth Kjeldgaard et al. In spite of the lack of a positive relationship for Ca. Oligotrophs SAR92 and Ca. The rRNA:rDNA to growth rate relationship varied among the isolates, even for those with the expected positive relationship. Kemp et al. A possible physiological explanation for this variation is that protein translation is more efficient in some taxa than in others owing to codon usage bias Andersson and Kurland, ; Novoa and Ribas de Pouplana, Differences in translational efficiencies lead to difference in ribosome numbers per cell Klumpp et al.
But these possible explanations do not address why some rRNA:rDNA versus growth rate relationships are not positive, which was the case for Ca. In contrast, Salter et al. We suspect that the complicated rRNA:rDNA-growth relationships seen for these isolates are caused in part from using data from the near-stationary phase. The physiological limitations that cause slow growth in the near-stationary phase are different from the limitations that are responsible for slow growth in the exponential phase of batch cultures.
The most common response of bacteria entering stationary phase, is to degrade rRNA Deutscher, However, there is evidence that some marine Vibrio may not immediately degrade their rRNA during the onset of starvation depending on the conditions inducing the stationary phase Kramer and Singleton, Ratios from oligotrophic Ca.
The number of ribosomes does not change greatly with growth state for Ca. Ratios for both oligotrophs varied much less than for the two copiotrophs examined here 5. Assuming that Ca. The discrepancy is resolved by using the ratio to growth relationship for Ca.
Using the Ca. In short, studies using 16S rRNA levels and other approaches both now indicate that growth rates of SAR11 are as high as or even higher than the average bacterium in marine waters.
To estimate the growth rates of SAR11 and the other three taxa in a natural community, we compared the rRNA:rDNA ratios measured in the culture experiments with ratios measured in bacterial communities along a transect in the Delaware estuary. This growth rate of Ca. Our results suggest that the type of Ca. This result contributes to the discussion of SAR11 survival strategy Zhao et al. Assuming that our results for this Ca. The other bacteria grew more slowly than Ca.
Slow growth may explain the low abundance of SAR92 in the estuary; it was found in only four of 24 samples. SAR92 does not appear to be adapted to the estuarine environment, probably reflecting that it was isolated from Pacific coastal waters Cho and Giovannoni, Growth rates of even the two copiotrophic bacteria appeared to be slower than growth of Ca.
Bacteria in the broader phylogenetic group Roseobacter , which contains R. Ratios for MED in natural communities of the Delaware were more than fold lower than ratios of MED growing in culture, suggesting slow growth in the estuary.
In fact, ratios in the estuary Previous studies indicate that some members of Flavobacteria , which includes MED, can grow at rates from 0. Although the copiotrophs we examined appeared to grow slowly in the Delaware, they are likely adapted to the estuary; both copiotrophs were present in over 20 of 24 samples, only slightly fewer than the apparently rapidly growing Ca.
The persistence of the two copiotrophs in the estuary may be owing to short bursts of growth that occurred at other times of the year when we did not sample or were not captured by our sampling approach. Copiotrophs likely grow rapidly when associated with particles and other patches where nutrients are temporarily replete Koch, ; Lauro et al.
Incidents of fast growth by copiotrophs may be captured by simply taking more standard large samples over time or by focusing sampling efforts on particles and other small scale patches Long and Azam, Of course growth rates and thus rRNA:rDNA ratios will vary greatly in the Delaware and elsewhere because of variation in potentially many biogeochemical properties, such as temperature and organic material. A key question is how variation in these properties affects the relationship between the ratios and growth and the extrapolation from laboratory experiments to natural environments.
Temperature in the Delaware estuary ranged from 9. In Salmonella, however, there is no effect of temperature on cellular rRNA content beyond the effect temperature has on the growth rate Schaechter et al. Another difference between our culture experiments and the growth of these organisms in the environment is the type and varying amount of carbon available for growth. Organic carbon sources used to grow bacteria in culture are less complex and more labile than those in the environment.
However, Schaechter et al. The rRNA:rDNA of the marine oligotrophic bacteria examined in this study differed from the ratios in marine and non-marine copiotrophs examined by previous studies and this study. The rRNA:rDNA of the two oligotrophic bacteria was lower than predicted from work with copiotrophic bacteria, which has implications both for understanding the physiology of these organisms and for measuring their growth rate in the environment. We suggest that ribosomes of oligotrophic bacteria are more efficient, which enables them to achieve faster growth with fewer ribosomes than seen for copiotrophic bacteria.
It may be evolutionarily advantageous for bacteria to have as few ribosomes as possible to succeed in the ocean and other oligotrophic environments, as ribosomes are costly to synthesize in terms of both rRNA and ribosomal proteins. The regulation of ribosomes has implications for using rRNA to explore growth rates in the environment. Ratios of rRNA:rDNA currently still seem the best option for determining growth rates of marine bacteria at a high phylogenetic resolution and for exploring the contribution of specific taxa to the carbon cycle.
Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59 : — Andersson S, Kurland C. Codon preferences in free-living microorganisms. Microbiol Rev 54 : — The ecological role of water-column microbes in the sea. Mar Ecol 10 : — Article Google Scholar. Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses. ISME J 7 : — Bremer H, Dennis P. Google Scholar. Global biogeography of SAR11 marine bacteria.
Mol Syst Biol 8 : 1— The ribose nucleic acid content and cell growth of Bact. J Chem Soc 1 : — Aquat Microb Ecol 57 : — Bacterial diversity, community structure and potential growth rates along an estuarine salinity gradient.
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