FEMS Microbiology Ecology 30 (1999) 253^260
Small ribosomal RNA content in marine Proteobacteria during
non-steady-state growth
Lee Kerkhof
a
a;
*, Paul Kemp
b
Institute of Marine and Coastal Sciences, 71 Dudley Rd. Cook College, Rutgers University, New Brunswick, NJ 08901-8521, USA
b
Marine Science Research Center, SUNY-Stony Brook, Stony Brook, NY 11794-5000, USA
Received 16 April 1999; revised 27 July 1999 ; accepted 27 July 1999
Abstract
Nine strains of marine Proteobacteria were assayed for nucleic acid content during non-steady-state growth to assess
whether a species-specific growth rate based on rRNA content is feasible for environmental samples. The large and small
ribosomal subunits and genomic DNA were quantified using image analysis. It was found that the maximal intracellular
concentration of 16S rRNA during batch growth for the bacteria averaged 155 fg þ 60 (S.D.) per cell for eight of the nine
marine bacteria in the exponential phase (with the exception of one strain, Pac 218). The dilution/decay of 16S rRNA/cell was
rapid with a return to pre-shift up values within 6^12 h for all strains except Vibrio fisherii. An overall relationship between the
RNA:DNA ratio and the specific growth rate for non-steady-state growth for all bacterial strains was not observed as
previously described for other Proteobacteria during steady-state growth. However, a predictable relationship between rRNA
content and growth rate for many isolates during batch growth was observed. Furthermore, the rapid kinetics of intracellular
rRNA levels indicates it will be feasible to assess whether specific bacteria are in steady state or non-steady state in the marine
environment. If the condition of steady state is met for a specific Proteobacterial group in an environmental sample, it will be
possible to estimate species-specific growth rates by measuring rRNA content. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Marine bacterium; Species-speci¢c growth rate assay; Non-steady-state growth; Ribosomal RNA content; RNA :DNA ratio
1. Introduction
Determining the in situ growth rate of micro-organisms is of fundamental importance in marine
ecology. Methods which depend on measuring the
concentrations of intracellular components [1] or incorporation of labelled precursors into cellular con-
* Corresponding author. Tel.: +1 (908) 932 6555;
Fax: +1 (908) 932 6520; E-mail: [email protected]
stituents [1^4] have produced major advances in our
understanding of the role of bacteria in marine ecosystems and are widely applied for addressing the
growth rates of natural populations of marine bacteria [5,6]. These whole population approaches estimate the average growth rate of the entire population of micro-organisms and are useful for
computing carbon £uxes and secondary production.
However, they cannot assess the role of di¡erent
species within the assemblage, or determine the relative contribution of various bacterial species to bio-
0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 9 ) 0 0 0 6 4 - 1
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L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
geochemical cycling under di¡erent conditions. Furthermore, we do not know whether it is reasonable
to expect that the entire population of marine bacteria is all growing at a uniform rate. To better
understand the ecology of a particular bacterial species in the marine environment, we need a means to
measure the growth rate of a single species of bacteria in a natural sample independent of the other
bacteria and eukaryotes present in that sample.
One approach to develop a species-speci¢c growth
rate assay for natural populations of marine bacteria
is based on measurement of ribosomal RNA
(rRNA), a cellular marker providing information
on both the genetic and the metabolic condition of
the cell. The relationship between rRNA content and
growth rate has become well established for enteric
bacteria, including Escherichia coli, Salmonella typhimurium and Aerobacter aerogenes cells under di¡erent growth conditions [7^9]. These and other studies
demonstrated that the rRNA content per cell varies
with growth rate in the bacteria examined, regardless
of the type of nutrients available [7,8,10]. Much of
the initial research focused on growth rates typical of
these enteric strains, i.e. doubling times in the order
of minutes (20^400 min). Later work extended the
range of growth rates exhibiting a relationship between rRNA content to very slow doubling times
typical of non-enteric bacteria in the environment,
in the order of hours (6^60 h) [11^13]. Evidence
has also been presented for a global relationship
(at least among Proteobacteria) between rRNA content and growth rate under steady-state conditions
throughout this entire range of doubling times
from 0.3^60 h [12,14].
The advent of in situ hybridization techniques [13^
15] for quantifying the rRNA content of speci¢c cells
ushered in a new era for growth rate estimation in
complex samples. Fluorescent labels and CCD cameras have allowed researchers to detect individual
cells [16] and estimate the ribosome content for approximating species-speci¢c growth rates in the environment. However, interpretation of the degree of
£uorescent signal when screening a natural sample
by in situ hybridization remains problematic. For
example, in the marine environment, many studies
have shown Proteobacteria to be dominant members
of the oceanic community [17^23]. Using in situ techniques, one can measure the amount of rRNA from
a Proteobacterium in an oceanic sample and calculate an rRNA/DNA ratio. From these data, it is only
possible to estimate the growth rate if we assume:
1. the global relationship between rRNA content
and growth rate applies to the Proteobacterium
and
2. the bacteria being monitored are in a steady state
of growth.
However, it could be that many bacteria in the
marine environment are not in steady state or may
be in starvation/survival mode [24]. Thus, application of steady-state relationships to estimate growth
rate under these conditions may be inaccurate. This
study was initiated to elucidate the variability in
rRNA content in marine bacteria under nonsteady-state (batch growth) conditions. One question
that is addressed is whether the global relationship
between rRNA content and growth rate observed in
Proteobacteria in steady state [12,14] is also observed
during non-steady-state growth.
2. Methods
2.1. Micro-organisms and culture conditions
Nine heterotrophic bacterial strains (collected
from the Black Sea, the Atlantic, and the Paci¢c
oceans) were tested for rRNA and DNA content
along a standard growth curve. All bacteria are
members of the Proteobacterial phyla based on 16S
rRNA sequence data [25] and the nearest GenBank
match for each isolate is presented in Table 1. For
the growth rate studies, 10-day-old cultures were reinoculated into fresh CP medium [26] diluted (1:20)
with aged seawater. Twenty time points were taken
along a 10-day growth curve (10 hourly time points
in lag/exponential phase; and 10 time points in stationary/death phase). All samples were stored frozen
at 380³C until extraction. Optical density (600 nm)
was measured and samples preserved in formalin for
DAPI counts [27]. A global correlation between OD
and direct counts for all strains sampled throughout
the di¡erent growth curves was observed (bacterial
numbers = 30.74+0.12Ulog(OD) r2 = 0.81; n = 45).
Cell numbers were calculated at each time point
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L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
from OD data using the above relationship for all
strains. Speci¢c growth rates were determined from
changes in the derived cell numbers at each time
point during the exponential phase. Thus, the growth
rate was calculated on an hourly basis for virtually
every data point. Only 2 or 3 data points out of
90 re£ect sampling times greater than 1 h.
2.2. Extraction of nucleic acids
Duplicate cell pellets (106 ^108 cells) were collected
for nucleic acid extraction for all bacteria tested with
the exception of strain DOE-8. A modi¢ed, phenolchloroform extraction protocol [12] was used to purify both RNA and DNA. This method has been
shown to quantitatively recover RNA over the range
of cell concentrations used in this study [12]. Slight
modi¢cations include: 75 Wl of 500 mM EDTA vs.
100 in the original method and total nucleic acids
being precipitated in ethanol with glycogen (40 Wg)
as a carrier. All nucleic acid pellets were re-suspended in DEPC-treated water in volumes of 10^
1000 Wl. rRNA was separated from DNA by agarose
gel electrophoresis before quanti¢cation. One percent
agarose mini gels were run in 1UTAE bu¡er at
150 V for 1 h to achieve separation of all nucleic
acid fractions.
2.3. Mass determination of ribosomal RNA and DNA
Nucleic acid masses were measured using ethidium
bromide £uorescence and image analysis. This technique allows for simultaneous separation and quantitation of 23S and 16S rRNA and genomic DNA
[28]. (Additionally, calculation of 23S rRNA/16S
rRNA or RNA:DNA ratios can be used to detect
variations in extraction during the course of the experiment.) The gels were stained in zip-lock bags
with ethidium bromide (4 Wg ml31 ) for 15 min with
gentle agitation. This procedure was found to minimize staining variability across the gel with coe¤cients of variation 6 5% for DNA bands of comparable mass (data not shown). A computer image of
the stained gel was captured and analyzed using the
Fotoanalyst system (Fotodyne, Hartland, WI, USA).
Fluorescence of speci¢c bands was determined by
creating a mapping image (copy) of each gel using
the sharpen (laplacian) transformation with an 8-pix-
255
el edge detection aperture. The area and pixel intensity of each band on the original image was integrated using the mapping image as a guide.
Standards of V HindIII DNA (Boehringer Mannheim, Indianapolis, IN, USA) and rRNA were run
on all gels. In order to achieve good separation of
the DNA standard, the V sample was loaded and
pre-run for 15 min prior to all other samples.
rRNA standards were created and calibrated using
total rRNA from six marine bacteria in early exponential phase growth. The combined 23S and 16S
rRNA species were gel puri¢ed using the RNAid
kit (Bio 101, Vista, CA, USA) and quanti¢ed by
UV absorbance on a model DU-600 spectrophotometer (Perkin Elmer, Foster City, CA, USA) assuming
a conversion of 40 Wg RNA ml31 = 1 OD unit. (The
OD260 /OD280 ratios of the puri¢ed rRNAs were between 1.9 and 2.0.) The rRNA mass standard was
prepared by calibrating a total RNA sample of Pseudomonas stutzeri Zobell using serial dilutions of the
puri¢ed 23S and 16S rRNA mass standards from the
six bacterial strains.
3. Results
Nucleic acids were extracted from samples collected throughout the entire experiment (10-day incubation). An example of a gel containing these extracts is shown in Fig. 1. Intact rRNA (low
molecular mass) and DNA (high molecular mass)
bands were observed for all samples throughout
the growth experiment. No signs of RNA or DNA
degradation were apparent, indicating uniform extraction of all nucleic acids. The mass standards of
DNA and RNA included on each gel were found to
exhibit linear relationships between nucleic acid mass
and ethidium £uorescence with r2 v 0.91 (92% of the
standards had r2 v 0.95) verifying the quantitative
nature and low variability of the image analysis
methodology.
Most bacterial strains grew to an optical density of
0.2^0.3 within 15 h of inoculation (Fig. 2). All
strains, with the exception of BS5 30A, were in stationary phase within 24 h. Using the £uorescence
and cell numbers data it was possible to calculate
16S rRNA concentration per cell during growth by
averaging the mass of two 16S rRNA bands and
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L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
(S.D.)/cell for eight of the nine marine strains tested.
One strain exhibited a much higher peak in 16S
rRNA content during early exponential phase
(1040 fg/cell for strain Pac 218; Fig. 2G). Dilution/
decay of 16S rRNA within the bacterial cells was
initiated in mid-exponential phase for most cultures
with the exception of V. ¢sherii (Fig. 2I). Rapid rates
of dilution/decay were found in most strains with a
return to pre-shift up rRNA intracellular concentrations by the onset of stationary phase (6^12 h; Fig.
2A^I).
The computer images also allowed the calculation
of 23S rRNA/16S rRNA ratios to test for variability
in extraction between samples during growth. These
ratios were determined by dividing the mass of the
rRNA species in each extraction (lane). The ratios
were then averaged and standard deviations were
calculated. Since the rRNAs are most likely co-transcribed [29], any deviations in the 23S/16S ratio
would indicate selective extraction of either large or
small rRNAs during the experiment. The results are
presented in Fig. 3. Large to small rRNA ratios
ranged from less than one to four with most bacterial strains having 23S/16S ratios between two and
three. However, for all strains (with the exception of
2563D) nearly constant 23S/16S ratios were observed
during the ¢rst 24 h of the experiment indicating
peaks in 16S rRNA per cell are accompanied by
peaks in 23S rRNA per cell. Small variability is
seen in most samples (with the exception of 2563D)
indicating the extraction procedure is robust and
highly reproducible during this time period. These
results suggest the observed peaks in 16S rRNA/
cell (Fig. 2) represent changes in intracellular concentrations and are not due to variations in extraction
of rRNA subunits during exponential phase growth.
The nearly constant 23S/16S ratios also indicate little
Fig. 1. Agarose gel of nucleic acids extracted during non-steadystate growth. Lanes A^K are death and stationary phase. Lanes
M^S are exponential and lag phase. Lane L is V molecular mass
marker and lanes T^X are rRNA standards.
dividing by the cell number at any particular time
point. The results of the nucleic acid measurements
for exponential phase growth are presented in Fig.
2A^H. All strains with the exception of Alcaligenes
faecalis homari (Fig. 2H) and Vibrio ¢sherii (Fig. 2I)
exhibited a sharp peak in 16S rRNA/cell during
exponential phase. The amount of 16S rRNA during
this peak in concentration averaged 155 fg þ 60
Table 1
Molecular characterization (16S rRNA sequence) of the isolates used in this study
Strain
Nearest GenBank match
Percent identity
Overlap
Reference
2593A
2563C
2563D
BS5 30A
BS5 40A
DOE-8
Pac 218
Vibrio splendidus
Vibrio carchariae
Aeromonas sp.
Mariana Trench clone 01
Pseudoalteromonas sp. ANT6
V. splendidus
Pele's Vent bacterium OTU 5
98.3
96.4
98.0
99.1
98.4
95.7
97.6
521/530
514/533
488/498
535/540
430/437
491/513
480/492
[35]
[35]
^
[33]
[18]
[35]
[22]
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L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
257
Fig. 2. Graphs of optical density (E), 16S rRNA/cell (S) through time for the following marine strains: (A) 2593A, (B) 2563 C, (C) 2563
D, (D) BS5 30A, (E) BS540A, (F) DOE-8, (G) Pac 218, (H) A. faecalis homari, (I) V. ¢sherii. Error bars indicate S.D.
degradation is occurring during extraction, since
more of the 16S rRNA would be degraded per unit
time than 23S and the ratio would change. However,
two strains (DOE-8 and Pac 218) exhibited 23S/16S
ratios less than one indicating excess 16S rRNA in
these particular bacteria or a systematic bias for the
small rRNA that is constant throughout the experiment.
Using the cell number data, it was possible to
determine speci¢c growth rate and compare with
16S rRNA/cell during the incubation (Fig. 4). Three
patterns were apparent in the data. One strain, BS5
30A, demonstrated a direct linear relationship between speci¢c growth rate and 16S rRNA/cell (BS5
30A r2 = 0.85). Most other bacteria in the study, represented by DOE-8, had the peak in speci¢c growth
rate before of after the peak in 16S rRNA/cell. Finally, one strain (V. ¢sherii) did not have an obvious
relationship between speci¢c growth rate and 16S
rRNA content.
We used similar plots to ascertain the time shift
necessary to superimpose the peak in speci¢c growth
rate and 16S rRNA content for the remaining bacteria used in the study. This alignment of the peaks
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L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
Fig. 3. Graphs of 23S/16S rRNA ratio for the following marine strains : (E) 2593A, (b) 2563 C, (O) 2563 D, (P) BS5 30A, (F) BS540A,
(a) DOE-8, (*) Pac 218, (S) A. faecalis homari, (R) V. ¢sherii. The dashed line represents the theoretical ratio of 2.0. Error bars indicate
S.D.
allows for comparison of rRNA content and speci¢c
growth rates. The data are summarized in Table 2. A
linear relationship between growth and 16S rRNA/
cell under non-steady-state conditions was only observed in three strains, BS5 30A, DOE-8 and Pac 218
(r2 = 0.80^0.95). Correlation coe¤cients ranged from
r2 = 0.21 to 0.95. The global 16S rRNA/cell vs.
growth rate relationship was not found to be signi¢cant (r2 = 0.199) under non-steady-state conditions.
Table 2
Correlation coe¤cients and time shifts necessary to align peak
16S rRNA/cell and speci¢c growth rates for all strains tested
Strain
Correlation coe¤cient
(r2 )
Time shift
(h)
DOE-8
BS5 30A
Pac 218
2563C
2563D
2593A
BS5 40A
A. faecalis homari
V. ¢sherii
0.95
0.85
0.80
0.72
0.64
0.54
0.47
0.21
0.04
+2
0
+1
+2
+1
+1
31
+1
31
Global
0.199
^
4. Discussion
Two important ¢ndings came out of this study.
Primarily, the data presented here con¢rm that interpretation of rRNA signals from environmental samples using either £uorescent in situ hybridizations or
probing of total RNA is not straightforward.
Although the range of peak intracellular concentrations of 16S rRNA varied by a factor of 2^4-fold for
most marine Proteobacteria at comparable portions
of the growth curve, one strain (Pac 218) was found
to contain signi¢cantly more 16S rRNA during exponential phase growth than the other marine
strains. If you compare the highest and lowest 16S
rRNA content per cell during early exponential
growth (1040 fg/cell for Pac 218; and 70 fg/cell for
A. faecalis homari), a 15-fold di¡erence is observed.
This data suggest that there may be a small number
of strains detected in the environment displaying
15-fold di¡erences in 16S rRNA intracellular concentration that are actually at the same numbers and
growing at the same rate as most other bacteria in
the sample. An important implication of this ¢nding
is that techniques that rely on estimating bacterial
abundance and distribution based on probing of total RNA with universal probes will be signi¢cantly
FEMSEC 1071 1-10-99
L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
Fig. 4. Graphs of 16S rRNA/cell (S) and speci¢c growth rate
(E) through time for the strains indicated. Error bars indicate
S.D.
in£uenced by those bacterial strains containing high
levels of 16S rRNA, such as Pac 218.
With respect to growth rate estimations from environmental samples using rRNA content, the interpretation of data will also be di¤cult. If speci¢c
populations of bacteria in the marine environment
are in steady state, growth rate can be reasonably
estimated using the Proteobacterial RNA :DNA vs.
growth rate relationship previously described [12,14]
259
However, during non-steady-state conditions, intracellular 16S rRNA levels were not found to be linearly related to growth rate for most of the strains
tested (Table 2). Hence, estimations of growth rate
for these non-steady-state population will not be as
accurate.
If species-speci¢c growth rates are to be precisely
determined, it will be necessary to ascertain whether
the bacterial group in the sample is in steady state.
Our results suggest that assessment of whether many
marine microorganisms (not some Vibrios) are in
steady state in the ocean can be done by sampling
through time and measuring large increases or decay
in 16S rRNA/cell. In contrast with previous reports
of slower decay or retention of ribosomes during
stationary phase for Desulfobacter lactus [30] and
Vibrio spp. [31,32], most marine strains in this study
demonstrated a return to pre-shift up levels in 16S
rRNA/cell within 6^12 h. Additionally, samples were
collected for 10 days after inoculation to assess
whether ribosomes were retained in non-growing
cells. No strain exhibited signi¢cant ribosome retention during stationary phase. Therefore, if the 16S
rRNA content per cell in a speci¢c microbial population in a natural sample was observed to remain
stable over a 3^6-h time period, the bacterial cell will
most likely be in steady state. If the steady-state
criteria are met, then universal steady-state relationships [14] between RNA:DNA ratios and growth
(i.e. RNA/DNA = 1.65+6.01 mu0:73 ; r2 = 0.85) can
be reasonably applied to estimate species-speci¢c
growth rates.
In conclusion, intracellular concentrations of 16S
rRNA were not found to be globally related to all
speci¢c growth rates in all marine bacteria tested
during non-steady-state growth in contrast with previous reports of a highly consistent global relationship for Proteobacteria in steady-state growth.
Although a global relationship for non-steady-state
growth was not found, it is highly likely that there is
a predictable relationship between RNA and growth
rate for each isolate, even though it may not be
revealed by a simple linear regression. Our data
show consistent patterns of peak rRNA concentration associated with maximal growth rates. Furthermore, since many of the Proteobacterial strains used
in this study are found in samples or 16S rRNA
clonal libraries ranging from Antarctica [18] to Ha-
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L. Kerkhof, P. Kemp / FEMS Microbiology Ecology 30 (1999) 253^260
waii [22] and the Mariana trench [33] to surface
waters, these results should be applicable to other
parts of the ocean. Finally, additional research is
warranted to determine whether other molecules
such as pre-16S mRNA can better predict growth
rate [34] on a species-speci¢c basis in natural samples
for bacteria experiencing non-steady-state growth.
Acknowledgements
The authors wish to thank the anonymous reviewers for their constructive comments. This research was supported in part by funds from IMCS
at Rutgers University, and NSF Grants OCE 9023858 and OCE 92-96230 to L.K. and Department
of Energy Grants AC02-76CH00016 and FG0296ER62186 to P.K.
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