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com/scientificreports

OPEN Identification of a comprehensive

set of transcriptional regulators
involved in the long-term
survivability of Escherichia coli in
soil
Soma Nakamoto1, Ikki Kobayashi1, Koichi Watanabe1, Takeru Kikuta1, Sousuke Imamura2
& Tomohiro Shimada1
Bacteria that typically do not thrive in soil can survive therein for long periods. While much research
has been conducted on the external environmental factors affecting the long-term survival of bacteria
in soil, their inherent factors are poorly understood. To adapt to environmental changes, bacteria alter
their gene expression patterns using transcriptional regulators such as sigma factors. Using Escherichia
coli as a model bacterium, we examined the effects of each transcriptional regulator on the long-term
survivability of E. coli in soil. The survivability of 294 E. coli strains deficient in transcriptional regulators
in soil was measured over 6 weeks. The results showed that ten strains deficient in transcription factors
significantly reduced survivability, whereas four deficient strains increased it. The functions common to
several of these transcriptional regulators included carbon and nitrogen metabolism, stationary phase
adaptation, and osmotic stress adaptation. These transcription factors are often global regulators
and conserved among other pathogenic bacterial species. Taken together, we successfully identified
a comprehensive set of transcription factors involved in the long-term survival of E. coli in soil. These
findings will be useful for understanding the mechanisms underlying the adaptation of microorganisms
to soil environments.

Keywords Long-term survivability in soil, Transcription factor, Sigma factor, Gene regulation, Escherichia
coli

Various bacterial species are present in the soil and reportedly survive for long periods. These bacteria provide
numerous ecosystem services through complex interactions between organisms within the soil and the soil itself;
however, some threaten human health1,2. Soil-borne bacterial infections are classified as being caused by either
soil-transmitted pathogens or euedaphic pathogenic organisms, with the former struggling to persist in the soil1.
Although Escherichia coli is considered to be predominantly a commensal of the gastrointestinal tract, recent
studies suggest that it is also capable of long-term survival and growth in environments outside the host. It is an
important indicator species reflecting potential environmental fecal contamination events3,4. Many studies have
focused on the survival of foodborne pathogens in soil, including Shiga toxin-producing E. coli O157:H7 (STEC)
and enterohemorrhagic E. coli O104:H4 (EHEC) for more than 1 month5–8. Several studies have researched the
environmental factors affecting the long-term viability of E. coli in soil, and factors such as soil pH9, moisture level,
temperature10, soil depth11, nutrients12, and cover crop species and season13 have been identified. While these
external environmental factors have been clarified, little is known about the internal factors of E. coli affecting its
long-term viability in soil. To date, the only factor whose involvement is understood is RpoS, a stationary phase
sigma factor that identifies promoters for transcribing genes involved in stationary phase adaptation and stress
response14,15. Previous studies have demonstrated that deletion of rpoS reduces the long-term survivability of
both pathogenic and nonpathogenic E. coli in soil8,16–19. However, no other genes involved in long-term survival
in soil have been identified. The inherent cues that E. coli detects and the genes it employs to enable long-term
survival in soil remain unclear. In addition, microorganisms other than model pathogenic microorganisms, such

1School of Agriculture, Meiji University, 1-1-1 Kawasaki-Shi, Tokyo, Kanagawa 214-8571, Japan. 2Space
Environment and Energy Laboratories, NTT Corporation, Musashino-Shi, Tokyo 180-8585, Japan. email:
[email protected]; [email protected]

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as E. coli and Salmonella, have not been studied in this regard, and the underlying molecular mechanisms are
poorly understood.
Escherichia coli is a model bacterium, and, as a single organism, has the most comprehensive collection of
genetic information and functions. It contains approximately 4,700 protein-coding sequences in its 4.6 Mbps
genome, seven sigma factors, and approximately 300 transcription factors to precisely regulate the genes required
to respond to various environmental changes14,20. Including RpoS, sigma factors are among the subunits of RNA
polymerase used to recognize promoters and alter the gene set used in responding to environmental changes14,21.
Transcription factors become active or inactive when they interact with effector molecules or are modified,
activating or repressing the transcription of target genes20,22. Transcription factors are categorized into four
groups based on the number of their target genes, each influencing genome regulation to varying extents –
nucleoid-associated regulators, which control approximately a thousand genes; global regulators, which control
hundreds of genes; local regulators, which control tens of genes; and single-target regulators, which controls
a single or few genes23–25. Since the functions and physiological roles of approximately four-fifths of the 300
transcription factors in E. coli have been identified26, a comprehensive identification of the transcriptional
regulators involved in long-term survival of E. coli in soil is the best research strategy to understand what bacteria
sense and the genes they utilize for long-term survival in soil.
In this study, we analyzed all of the transcriptional regulators in E. coli and the effect on long-term survival
of the bacteria in soil when one of them is deleted. Furthermore, by analyzing the functions and physiological
roles of the affected E. coli transcriptional regulators and their conservation among bacterial species, we propose
a mechanism for long-term survival of the bacteria in soil.

Results
Assay for the long-term survival of E. coli wild-type strain BW25113 in soil
The soil viability of wild-type E. coli BW25113, a derivative of the E. coli K-12 strain, fluctuated under the
conditions of this experiment. Approximately 2 × 107 E. coli cells mixed in 1 g of soil were incubated in a constant
temperature and humidity chamber at 25 °C and 60% humidity. A common method for determining the number
of viable cells in soil is quantifying the amount of genomic DNA using the polymerase chain reaction method.
However, this method does not distinguish between living and dead cells. Thus, we used the diluted platelet
culture method to count the number of viable cells as the number of colony-forming units on an agar plate at
0, 3, 7, 21, and 42 days (Fig. 1A). Although LB liquid medium was used for pre-culture, E. coli growth media
reportedly does not influence its survival in soil under static conditions27. As a result, the viable cells of the E. coli
wild-type strain decreased from 2.2 × 107 at 0 days to 1.6 × 106, 9.5 × 105, and 2.5 × 105, reaching 5.9 × 104 after 3, 7,
21, and 42 days (Fig. 1B). Three samples were used for each sampling point, and the wild-type strain experiment
was verified for reproducibility by conducting the experiment ten times independently. In these experiments,
the results for each experiment varied within a range of ± twofold of the mean, and the experimental error was
estimated to be approximately fourfold of the mean. The variations in viable cell counts from days 0 to 3, 7,
21, and 42, converted to percentages, were 100, 7.4, 4.3, 1.1, and 0.27%, respectively. By day 7, the counts had

Fig. 1. Overview of the long-term soil survival assay system and observations of Escherichia coli wild-type
strain. (a) Schematic of the long-term soil survival assay. (b) Fluctuations in the survival of E. coli wild-type
strain under this experimental system. The average colony-forming units (CFUs) per gram of soil are indicated
by closed black circles. Survivability normalized to the initial observation point is indicated by white circles.
Error bars represent the standard deviation of ten independent experiments.

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dropped to less than one-twentieth, and the pattern of gradual decline thereafter was consistent with previous
reports27.

Effects of transcription factor deletion on the long-term survivability of E. coli in soil and its
evaluation criteria
To determine the inherent cues detected by E. coli and the genes it utilizes for long-term survival in soil, we
comprehensively analyzed the survival of transcription and sigma factor-deficient E. coli in soil. We focused on
seven sigma factors and approximately 300 transcription factors of E. coli K-1214,20, which are based on the list
of E. coli K-12 transcriptional regulators previously reported by Ishihama and Shimada28. Among these, 290
transcription factor-deficient strains and four sigma factor-deficient strains from the KO collection, a single-
gene disruption collection of E. coli, were included in the analysis29. The survival rates for all 294 transcription
regulator-deficient strains were observed at 0, 3, 7, 21, and 42 days, as in the wild-type strains. To confirm
reproducibility, two sets of experiments were performed independently, with three biological independent
samples for each observation point. As the actual assay examined deficient strains for every several to a dozen
strains, the assay was always performed alongside the wild-type strain to internally correct for experimental
errors. As a result, the error for wild-type strains was within a fourfold range in all experimental groups (Fig. 1B).
To facilitate the evaluation of the effect of transcriptional regulator deficiency, the survivability of each
deficient strain was evaluated by setting the colony-forming units (CFUs) on day 0 as 100%, and the CFUs over
time were evaluated relative to the survival rate in the wild-type strain. The survival results for all strains in soil
are presented in Table S1. Similar to the wild-type strain, the number of viable cells in all strains decreased as the
number of days passed after mixing with the soil. The effect of the loss of transcriptional regulators also became
greater as the days passed. Most defective strains had survival rates comparable to those of the wild-type strains,
but some strains had decreased or increased survival rates. Ten strains showed a 100-fold or greater decrease
in survivability after 42 days. In contrast, under the same conditions, only a few strains exhibited increased
survivability, and this increase was approximately tenfold. As the experimental error between each experiment
was within fourfold of the wild-type strain, the cutoff levels determined to be significant in experimental
sets 1 and 2 were set at tenfold for a decrease in survivability and fivefold for an increase. Furthermore, for
deficient strains that were severely affected by transcription factor deletion, complementation experiments
were conducted to confirm the effect of the deficiency by complementing transcription factor induction with a
plasmid transformant.

Transcription factors whose deletion reduced long-term survival of E. coli in soil

The deficiency of ten transcriptional regulators resulted in a tenfold or greater reduction in survival compared
to that in wild-type strains in two independent experiments at 42 days (Fig. 2, KO-1 and KO-2). The effects of
these transcriptional regulator deficiencies were further confirmed by conducting a third experiment. Because
the effect of transcriptional regulator deficiency on survival increased with the passage of days, the survival
rates in this third experiment were measured at days 0, 21, and 42. As a result, the same level of influence
was observed for all ten transcriptional regulators (Fig. 2A, KO-3), among which two were sigma factors and
eight were transcription factors (Table 1A). For the ten transcriptional regulator-deficient strains for which
experimental reproducibility was confirmed, further plasmid complementation experiments were performed to
verify the effect of these transcription factors. Observations of viability after 42 days showed that ΔihfA, ΔrpoS,
and ΔdinJ strains recovered their viability to almost the same levels as those of the wild-type strain following
plasmid complementation (Fig. 2B as ns on each bar, Table 1A). ΔabgR partially recovered viability, and ΔrpoN,
ΔygbI, Δdps, ΔompR, Δmlc, and Δlrp showed little to no change.
Several previous studies have indicated that in the long-term survival of either pathogenic or nonpathogenic
E. coli in soil, deletion of rpoS, which recognizes the promoters of stationary phase and stress-response genes,
significantly reduces long-term survivability8,12,18. The results for ΔrpoS were in agreement with those of
Somorin et al.18, who used the same strain, BW25113, and its rpoS-deficient strain and found that the difference
in survival rate increased with time. In addition to rpoS, whose effect is known, rpoN, which encodes the RpoN
sigma factor required for the promoter recognition of a group of nitrogen-related genes, was newly identified as
being required for long-term survival in soil.
The eight transcription factors required for long-term survivability in soil included abgR, dinJ, dps, ihfA, lrp,
mlc, ompR, and ygbI (Table 1A, Fig. 2). Deletion of abgR or ihfA had a significant effect in all experiments, with
survival in soil at 42 days being less than 1% of that of the wild-type strain. ΔrpoN showed a marked decrease
in survival from day 21 under the condition, indicating an early effect. The other effects were observed over
relatively long periods, extending up to day 21 or 42 (Fig. 2A).

Transcription factors whose deletion increased the long-term survival of E. coli in soil
The deficiency of four transcription factors, nhaR, cbl, cadC, and crp, resulted in a fivefold or greater increase in
survival compared to that of the wild-type strains in two independent experiments at 42 days (Fig. 3A). A third
experiment was also conducted, and the reproducibility of the effects was confirmed in all four deletion strains.
The increase in long-term survival in soil for strains deficient in these transcription factors was approximately
5- to tenfold compared to that of the wild-type strain. The effect of crp deficiency appeared from 3 days of
incubation, whereas those of nhaR, cbl, and cadC occurred after 21 days. Furthermore, complementation
experiments with expression vectors were performed. The results showed that ΔnhaR had a lower survivability
than that of the wild-type strain, and crp complementation reduced the survivability of Δcrp to almost the same
levels as those of the wild-type strain (Fig. 3B, Table 1B). In the case of Δcbl and ΔcadC, both expression plasmids
showed little complementation effects.

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Fig. 2. Transcriptional regulators whose deletion reduced the long-term survival of Escherichia coli in soil. (a)
Among the approximately 300 transcriptional regulators of E. coli (Table S1), the deficiency of ten regulators
reduced long-term survival in soil at 25 °C and 60% humidity. Sampling was performed on days 0, 3, 7, 21, and
42, with three independent replicates each. Standard deviations are indicated by error bars. The CFUs on day
0 were taken as 100%, and CFUs for each sampling time were compared for survivability and plotted on the
graph. Time is shown in days (d) on the x-axis, and survivability is shown as a percentage on the y-axis. The
black color shows the results of wild-type strain BW25113 (WT), the red color shows the results of the first set
experiment for the deficient strain (KO-1), and the blue color shows the results of the second set experiment
for the deficient strain (KO-2). The third set experiment on the deficient strain was performed on days 0,
21, and 42 and is shown in green (KO-3). (b) Forty-two-day survival of transcription factor-deficient strains
compared to the wild-type strain. Red, blue, and green bars indicate the results of the first (KO-1), second
(KO-2), and third experiments (KO-3), respectively. The yellow bars indicate the results of the first experiment
(C-1), and the orange bars indicate the results of the second experiment (C-2) when the defective strain was
complemented with the target transcriptional regulator using an expression vector. Error bars represent the
standard deviation of three independent biological replicates. Statistical significant differences between the
wild-type strain and the deficient strain or complementary strain in each experiment were obtained using
Student’s t-test with two-tailed test and are represented by asterisks (**p < 0.01, *p < 0.05, ns: Not significant)
above the bar. Statistically significant differences between three experiments with the wild-type strain and the
deficient strain and two experiments with the wild-type strain and the deficient strain were also determined
by Student’s t-test with two-tailed test and are indicated with lines and asterisks (**p < 0.01, *p < 0.05, ns: Not
significant).

Discussion
By comprehensively analyzing the transcriptional regulators of E. coli, which has the largest accumulation of
functional information on the genes of a single organism, we aimed to identify the transcriptional regulators
that sense and control the mechanisms of long-term microorganism survival in the soil. The transcriptional
regulators of E. coli comprise seven sigma factors for recognizing promoters and approximately 300 transcription
factors that regulate transcription levels14,20,21. We evaluated the long-term soil viability of 294 transcription
regulator-deficient strains in the KO collection—a collection of single-gene-disruption strains of E. coli—and
identified 14 affected transcription regulators. These included the sigma factor rpoS, which has previously been
reported as being important for maintaining the long-term survivability of E. coli in soil8,12,18. rpoS is the only

Scientific Reports | (2025) 15:4279 | https://doi.org/10.1038/s41598-025-85609-8 4

 Table 1. List of transcriptional regulators affecting the long-term survival of Escherichia coli in soil. Compared to the wild-type strain. Italics indicates a tenfold or more reduction in long-term
survival in soil for transcription factor-deficient strains, while underline indicates a fivefold or more increase.

Scientific Reports |
Survival rate (Ratio to wild−type strain)
Experiment-1 Experiment-2

Transcr- Altern-
iptional ative
regulator Strain Naming Effector names Family 0 days SD 3 days SD 7 days SD 21 days SD 42 days SD 0 days SD 3 days SD 7 days SD 21 days SD 42 days SD
(A) Transcription factors whose deficiency reduced E. coli survival
Regulator of

(2025) 15:4279
aminobenzoyl
AbgR JW1333 YdaK LysR 1.0.E + 00 7.1.E−02 2.8.E−01 1.4.E−01 1.1.E−01 7.9.E−02 1.3.E−02 1.1.E−02 2.6.E−03 1.8.E−02 1.0.E + 00 6.5.E−01 4.2.E−01 2.1.E−01 1.7.E−01 1.2.E−01 1.4.E−02 6.6.E−03 2.1.E−03 7.6.E−03
glutamate
utilization
Hid,
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Integration host
IhfA JW1702 HimA, HU 1.0.E + 00 1.6.E−01 7.0.E−01 8.7.E−01 5.5.E−01 1.2.E + 00 1.0.E + 00 3.0.E + 00 4.1.E−03 1.4.E−02 1.0.E + 00 1.5.E−01 2.6.E−01 4.3.E−02 4.5.E−02 4.3.E−02 1.2.E−01 2.7.E−02 6.5.E−03 1.8.E−02
factor A
IHF−a
RNA
polymerase
GlnF,
RpoN* JW3169 sigma subunit – 1.0.E + 00 4.1.E−01 2.3.E + 00 3.0.E + 00 6.6.E−01 7.9.E−01 3.6.E−04 1.1.E−03 1.5.E−03 1.7.E−04 1.0.E + 00 2.3.E−01 2.4.E−01 1.9.E−01 1.4.E−01 7.2.E−02 1.3.E−02 3.1.E−03 2.2.E−02 4.7.E−03
NtrA
for nitrogen
related genes
RNA
polymerase
sigma subunit
RpoS* JW5437 KatF – 1.0.E + 00 1.4.E−01 4.3.E−01 1.7.E−01 4.1.E−02 1.5.E−02 7.2.E−03 5.2.E−03 1.3.E−02 8.9.E−03 1.0.E + 00 3.8.E−01 1.8.E−01 1.3.E−01 2.3.E−01 1.1.E−02 4.7.E−02 4.1.E−02 3.9.E−02 5.1.E−02
for stationary
phase related
genes
YgbI JW2705 Unknown DeoR 1.0.E + 00 2.6.E−01 5.8.E−01 4.0.E−01 4.8.E−01 2.6.E−01 2.4.E−01 1.2.E−01 1.9.E−02 1.3.E−02 1.0.E + 00 4.8.E−02 4.8.E−01 1.0.E−01 1.4.E + 00 3.0.E−01 4.9.E−01 8.2.E−02 3.8.E−02 1.1.E−02
DNA damage-
DinJ JW0216 inducible SosA Xre 1.0.E + 00 1.1.E−01 3.5.E−01 1.8.E−01 6.6.E−01 4.0.E−01 2.5.E−03 1.9.E−03 1.1.E−05 7.2.E−03 1.0.E + 00 2.5.E−01 2.7.E + 00 1.4.E + 00 6.5.E−01 3.4.E−01 6.9.E−02 1.9.E−01 6.8.E−02 4.2.E−02
protein J
DNA protection
PexB,
Dps JW0797 during Dps 1.0.E + 00 1.2.E−01 6.3.E−01 9.7.E−02 2.6.E−01 1.8.E−01 3.5.E−01 2.4.E−01 9.5.E−02 3.4.E−02 1.0.E + 00 1.0.E−01 7.6.E−01 1.2.E−01 3.6.E−01 3.9.E−02 6.1.E−01 2.0.E−01 3.8.E−02 9.6.E−03
Vtm
starvation
Outer Acetyl−
Cry,Kmt,
OmpR JW3368 membrane Phos- OmpR 1.0.E + 00 4.6.E−02 3.0.E−01 1.3.E−01 3.2.E−01 1.1.E−01 3.2.E−01 3.4.E−01 4.7.E−02 3.5.E−02 1.0.E + 00 2.3.E−01 1.9.E + 00 5.4.E−01 1.6.E + 00 1.7.E−01 1.6.E + 00 7.2.E−01 6.0.E−02 1.3.E−02
OmpB
protein regulator phate

| https://doi.org/10.1038/s41598-025-85609-8
Regulator to
Mlc JW1586 make large DgsA NagC 1.0.E + 00 1.6.E−01 3.1.E + 00 6.6.E−01 3.6.E−01 2.8.E−01 8.2.E−01 2.0.E + 00 3.7.E−02 5.4.E−02 1.0.E + 00 4.6.E−01 4.5.E−02 1.3.E−01 1.0.E−01 9.2.E−02 1.4.E−01 1.0.E−01 9.5.E−02 6.0.E−02
conolies
Leucine- AlsB,LblA,
responsive LivR,LstR,
Lrp JW0872 Leucine AsnC 1.0.E + 00 9.2.E−02 5.0.E−01 2.0.E−01 4.8.E−01 3.4.E−01 3.3.E−01 3.6.E−01 5.9.E−02 4.4.E−02 1.0.E + 00 4.3.E−01 1.2.E + 00 4.9.E−01 1.9.E + 00 1.5.E + 00 7.0.E−01 6.4.E−02 8.1.E−02 3.9.E−02
regulatory Mbf,OppI,
protein RblA
*Sigma factor
(B) Transcription factors whose deficiency increased E.coli survival
Na+/H+
NhaR JW0019 antiporter Na+ AntO LysR 1.0.E + 00 2.2.E−01 2.5.E + 00 3.0.E + 00 2.3.E + 00 2.0.E + 00 9.9.E + 00 5.8.E + 00 2.0.E + 01 1.1.E + 01 1.0.E + 00 2.5.E−02 1.1.E + 00 6.4.E−02 1.2.E + 00 1.3.E−01 3.9.E + 00 1.1.E + 00 6.3.E + 00 1.6.E + 00
regulator
CysB−like Adeno-
regulator sine
Cbl JW1966 of aliphatic 5′− LysR 1.0.E + 00 3.7.E−02 1.5.E + 00 5.5.E−01 1.2.E + 00 8.7.E−01 3.0.E + 00 2.6.E + 00 1.1.E + 01 4.8.E + 00 1.0.E + 00 1.4.E−01 1.3.E + 00 2.5.E−01 1.7.E + 00 4.8.E−01 1.7.E + 00 3.5.E−01 6.0.E + 00 1.7.E + 00
sulfonate phospo-
utilization sulfate
Activator of
CadC JW4094 cadverine LysP CadC 1.0.E + 00 2.8.E−02 3.0.E + 00 6.0.E−01 2.9.E + 00 1.2.E + 00 8.2.E + 00 3.4.E + 00 1.2.E + 01 3.9.E + 00 1.0.E + 00 1.3.E + 00 2.5.E + 00 1.3.E + 00 2.2.E + 00 1.2.E + 00 3.6.E + 00 3.0.E−01 6.3.E + 00 1.6.E + 00
synthesis
cAMP receptor
Crp JW5702 cAMP Cap,Csm Crp 1.0.E + 00 2.1.E−01 1.4.E + 01 6.0.E−01 1.2.E + 01 1.1.E + 00 7.3.E + 00 3.8.E + 00 5.2.E + 00 9.7.E−01 1.0.E + 00 3.0.E−01 3.6.E + 00 2.1.E−01 4.1.E + 00 8.6.E−01 1.9.E + 00 7.0.E−01 6.2.E + 00 3.0.E + 00
protein

5
Scientific Reports |
Table 1. (Continued)
Survival rate (Ratio to wild−type strain)
Experiment-3 Complementary experiment-1 Complementary experiment-2
Transcri-
ptional Alternative

(2025) 15:4279
regulator Strain Naming Effector names Family 0 days SD 21 days SD 42 days SD Plasmid 0 days SD 42 days SD 0 days SD 42 days SD
(A) Transcription factors whose deficiency reduced E. coli survival
Regulator of
AbgR JW1333 aminobenzoyl YdaK LysR 1.0.E + 00 2.3.E−03 5.2.E−02 1.0.E + 00 9.7.E−02
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1.8.E−01 2.0.E−01 2.3.E−01 1.1.E−03 JW1333-AM 1.0.E + 00 9.2.E−01 3.5.E−02 6.0.E−02 1.1.E−01
glutamate utilization
Integration host Hid,HimA,
IhfA JW1702 HU 1.0.E + 00 1.7.E−01 4.5.E−01 4.3.E−02 2.5.E−03 7.9.E−03 JW1702-AM 1.0.E + 00 1.3.E + 00 2.7.E−01 1.7.E−01 1.0.E + 00 1.6.E−01 3.0.E−01 4.4.E−01
factor A IHF−a
RNA polymerase
sigma subunit for
RpoN* JW3169 GlnF, NtrA – 1.0.E + 00 3.6.E−01 4.3.E−02 2.1.E−02 2.0.E−02 1.6.E−02 JW3169-AM 1.0.E + 00 9.3.E−01 3.9.E−01 2.2.E−01 1.0.E + 00 1.7.E−02 9.3.E−02 1.1.E−01
nitrogen related
genes
RNA polymerase
sigma subunit for
RpoS* JW5437 KatF – 1.0.E + 00 4.1.E−01 1.9.E−01 2.6.E−01 1.7.E−02 1.9.E−02 JW5437-AM 1.0.E + 00 6.1.E−01 7.3.E−01 2.4.E−01 1.0.E + 00 2.9.E−01 3.4.E−01 1.3.E−01
stationary phase
related genes
YgbI JW2705 Unknown DeoR 1.0.E + 00 1.5.E−01 5.5.E−01 7.9.E−02 7.4.E−03 1.4.E−02 JW2705-AM 1.0.E + 00 5.9.E−01 9.6.E−02 6.8.E−02 1.0.E + 00 2.4.E−01 3.5.E−01 2.5.E−01
DNA damage-
DinJ JW0216 SosA Xre 1.0.E + 00 2.9.E−01 4.1.E−01 3.7.E−02 1.2.E−02 1.8.E−02 JW0216-AM 1.0.E + 00 6.6.E−01 1.8.E + 00 1.3.E + 00 1.0.E + 00 5.9.E−01 4.5.E + 00 4.8.E + 00
inducible protein J
DNA protection
Dps JW0797 PexB,Vtm Dps 1.0.E + 00 1.3.E−01 1.1.E + 00 1.2.E−01 1.7.E−02 4.0.E−02 JW0797-AM 1.0.E + 00 4.2.E−01 4.5.E−02 2.7.E−03 1.0.E + 00 4.1.E−01 3.6.E−02 3.7.E−02
during starvation
Acetyl−
Outer membrane Cry,Kmt,
OmpR JW3368 Phos- OmpR 1.0.E + 00 2.2.E−01 2.3.E−01 8.6.E−01 9.5.E−02 3.9.E−02 JW3368-AM 1.0.E + 00 6.2.E−01 1.5.E−01 1.1.E−02 1.0.E + 00 5.0.E−01 3.2.E−01 1.5.E−01
protein regulator OmpB

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phate
Regulator to make
Mlc JW1586 DgsA NagC 1.0.E + 00 1.6.E−01 3.9.E−01 6.4.E−01 7.6.E−02 4.3.E−03 JW1586-AM 1.0.E + 00 9.3.E−01 6.9.E−02 9.2.E−02 1.0.E + 00 5.5.E−01 5.5.E−02 6.2.E−02
large conolies
AlsB,LblA,
Leucine-responsive LivR,LstR,
Lrp JW0872 Leucine AsnC 1.0.E + 00 3.1.E−01 7.5.E−02 3.7.E−02 7.7.E−02 8.2.E−02 JW0872-AM 1.0.E + 00 6.3.E−01 3.7.E−01 2.3.E−01 1.0.E + 00 5.9.E−01 1.3.E−01 1.5.E−01
regulatory protein Mbf,OppI,
RblA
*Sigma factor
(B) Transcription factors whose deficiency increased E.coli survival
Na+/H+ antiporter
NhaR JW0019 Na+ AntO LysR 1.0.E + 00 1.2.E−01 2.8.E + 00 2.9.E−01 7.6.E + 00 2.2.E + 00 JW0019-AM 1.0.E + 00 5.7.E−01 1.7.E−01 1.0.E−01 1.0.E + 00 2.6.E−01 1.0.E−01 1.1.E−01
regulator
Adeno-
CysB−like regulator
sine 5′−
Cbl JW1966 of aliphatic sulfonate LysR 1.0.E + 00 2.6.E−02 5.5.E + 00 1.5.E + 00 8.6.E + 00 3.1.E + 00 JW1966-AM 1.0.E + 00 8.6.E−01 4.6.E + 00 2.0.E + 00 1.0.E + 00 5.8.E−01 3.7.E + 00 6.8.E−01
phospo-
utilization
sulfate
Activator of
CadC JW4094 LysP CadC 1.0.E + 00 1.4.E−01 4.5.E + 00 1.9.E + 00 5.6.E + 00 1.1.E + 00 JW4094-AM 1.0.E + 00 3.4.E−01 6.7.E + 00 3.3.E + 00 1.0.E + 00 4.7.E−01 4.4.E + 00 6.0.E + 00
cadverine synthesis
cAMP receptor
Crp JW5702 cAMP Cap,Csm Crp 1.0.E + 00 7.1.E−02 2.0.E + 00 1.9.E−01 5.7.E + 00 3.3.E + 00 JW5702-AM 1.0.E + 00 4.2.E−01 8.8.E−01 4.5.E−01 1.0.E + 00 2.8.E−01 8.0.E−01 4.8.E−01
protein

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Table 2. Conservation of transcription factors in soil and environmental pathogenic bacteria affecting the long-term survivability of Escherichia coli in soil. Gene conservation was analyzed using
QuickBLASTP (target percent identity = ≥ 50%) based on the amino acid sequence. Bold indicates conserved; underline indicates not conserved. The bold and italics, underline of the transcription
regulator name indicates comparison with the wild-type strain. Italics indicates a reduction in long-term survival in soil for strains deficient in the transcription regulator. Bold indicates an increase.
Strains list from, *European Commission Joint Research Centre—Soil Borne Human Diseases. **World Health Organization—Global Priority Pathogens List.
JRC SBHD*
WHO GPPL**
Escherichia
coli str. Pseu- Acine- Strepto-
TF or K-12 Shigella Yersinia domonas Klebsiella Escheri- tobacter Staphy- Entero- Haemo- coccus
sigma substr. Eschericia dysente- Salmonel- enterocol- aerugi- Entero- Shigella pneumo- Salmonel- Serratia Providen- chia coli bauman- Morganel- Proteus lococcus coccus philus Helicobac- pneumo-
name Type W3110 coli riae la enterica itica nosa bacter spp spp. niae la spp. spp. cia spp. O157 nii la spp. spp. aureus faecium influenzae ter pylori niae
IhfA Nucleoid 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 0 0 1 0
Lrp Global 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 1 0 0
CRP Global 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 1 0 0

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OmpR Local 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 0 0 0
Dps Nucleoid 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 0 0 0
NhaR Local 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 0 1 0 0 0 0
Single-
AbgR 1 1 1 1 0 1 1 1 1 1 0 1 1 0 0 0 0 0 0 0 1
target
Single-
DinJ 1 1 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 0 0 0 0
target
Sigma
RpoS 1 1 1 1 1 0 1 1 1 1 1 0 1 0 0 0 0 0 0 0 0
factor
Sigma
RpoN 1 1 1 1 0 0 1 1 1 1 0 0 1 0 0 0 0 0 0 0 0
factor
Mlc Local 1 1 1 1 0 0 1 1 1 1 0 0 1 0 0 0 0 0 0 0 0
Single-
YgbI 1 1 1 1 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0
target
Single-
CadC 1 1 1 0 0 1 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0
target
Single-
Cbl 1 1 1 0 0 0 1 1 1 0 0 0 1 0 0 0 0 0 0 0 0
target

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Fig. 3. A transcriptional regulator whose deficiency increased the long-term survival of Escherichia coli in
soil. (a) The deficiency of four transcription factors of E. coli increased long-term survival in soil. (b) Forty-
two-day survival of transcription factor-deficient strains compared with that of the wild-type strain. The figure
description is the same as the legend for Fig. 2.

gene previously known to be important for microbial long-term survivability in soil, while the other 13 genes
were identified for the first time in this study (Table 1).
The role of transcriptional regulators in regulating genomic transcription is assessed by the number of
regulatory target genes. Transcription factors are classified by the number of their target genes23–25. Accordingly,
the classification of the 14 transcriptional regulators identified in the present study is shown in Fig. 4. In addition,
the physiological roles of the transcriptional regulators were classified based on the functions of their target
genes.
RpoS and Dps are transcriptional regulators involved in stationary phase adaptation. Upon entry into the
stationary phase, the genome expression pattern is markedly altered by downregulating growth-related genes
and upregulating stress-response genes. The stationary phase-specific minor sigma σ38, encoded by the rpoS
gene, is involved in switching the transcription pattern30. More than 100 RpoS promoters that can be recognized
by the RpoS RNA polymerase holoenzyme have been identified15 and reported to be essential for the long-term
survival of E. coli in soil, as described above. Dps is a highly abundant protein in stationary-phase E. coli and is
required for the multiple starvation response31,32 and viability in liquid culture for approximately 10 days33. Dps
is a universally conserved prokaryotic ferritin that, in many species, also binds DNA and protects against DNA-
damaging agents34. These findings suggest that the long-term survival of E. coli in soil requires large-scale gene
expression and a compact nucleoid structure, both of which characterize the stationary phase.
RpoN and Lrp are transcriptional regulators involved in nitrogen metabolism. The sigma subunit σ54, encoded
by the rpoN gene, recognizes the promoters of more than 100 genes and is involved in various nitrogen-related
functions24,25,35. As such, RpoN is involved in nitrogen stress and stringent responses36. Recently, the RpoN RNA
polymerase holoenzyme was proposed to function as a repressor for preventing the transcription of a growth
gene set24,25. Lrp is a global regulator for genes involved in transport, biosynthesis, catabolism, and amino acid
utilization in E. coli37–39. This suggests that the long-term survival of E. coli in soil also requires large-scale
expression of amino acids and other nitrogen-related genes, as well as the suppression of growth-related genes.
CRP and Mlc are transcription factors involved in carbon metabolism. CRP is a global regulator of genes
involved in carbon source utilization in the absence of glucose40, reportedly regulating hundreds of genes involved
in carbon source metabolism41,42. Activation of the rmf gene, which encodes RMF, leads to the formation of the
100S ribosome, thereby inactivating translation activity43. Mlc is a local regulator that controls the expression
of several genes encoding enzymes involved in carbohydrate metabolism, including the phosphotransferase and
phosphoenolpyruvate systems44,45, and deficiency has been reported to cause colony size enlargement46. The
transcription of mlc itself is reportedly regulated by CRP47. Collectively, these observations suggest that the long-
term survival of E. coli in soil requires large-scale gene regulation of carbon metabolism.
NhaR and OmpR are local regulators for osmotic pressure. The activity of NhaR is dependent on Na+ and
regulates the transcription of genes involved in adaptation to Na+ and alkaline pH48, ribosome biogenesis,
and stress conditions such as salt stress49. NhaR activates osmC, which encodes an osmotically inducible
peroxiredoxin50. OmpR regulates the transcriptional expression of more than 30 genes involved in adapting to

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Fig. 4. Transcriptional regulators affecting the long-term survival of Escherichia coli in soil. Transcriptional
regulators that affect the long-term viability of E. coli in soil were classified by the number of targets and their
function. Blue indicates a transcriptional regulator whose deletion decreases survivability, and red indicates a
transcriptional regulator that increases survivability.

changes in osmolarity, including major outer membrane porins38,39,51,52. These findings indicate that the long-
term survival of E. coli in soil requires large-scale gene regulation for osmolarity stress.
Taken together, these results suggest that appropriate responses to the stationary phase, nitrogen and carbon
metabolism, and osmotic pressure are required for the long-term survival of E. coli in soil (Fig. 4). As noted
above, the identified transcription factors regulate several genes and are common physiological phenomena that
seem to be shared among microbial species.
In the case of Dps and CRP, it may be important to repress the expression of unnecessary genes because of the
associated energy consumption of expressing a large gene set. dps, whose loss reduces survivability, suppresses
the expression of genes necessary for growth by compacting DNA in the stationary phase53, whereas crp, whose
loss increases survivability, activates genes related to carbon source uptake and metabolism in the absence of
phosphoenolpyruvate: sugar phosphotransferase system (PTS) sugars40, despite the lack of a carbon source
under these assay conditions. The effect on long-term survival of the bacteria in soil could be attributed to the
deletion of dps, which consumed energy by failing to repress a set of genes that should have been repressed,
and the deletion of crp, which consumed little energy by failing to activate a set of genes that would have been
activated.
AbgR has been suggested to regulate the abgABT operon involved in p-aminobenzoyl-glutamate utilization54,
while CadC activates the cadBA operon involved in the lysine-dependent acid resistance system55. Cbl
activates the tauABCD and ssuEADCB operons involved in aliphatic sulfonate utilization and sulfate starvation
homeostasis56. Meanwhile, DinJ represses the dinJ-yafQ operon involved in the toxin-antitoxin system57 and
cspE, a transcription antiterminator and regulator of RNA58. YgbI regulates the ygbJK operon, whose function
is unknown59. To date, these five transcription factors have been suggested to be single-target regulators that
control one or two targets in the E. coli genome and, thus, may have specific functions. Future research is needed
to elucidate the relationship between the function of each target gene and its long-term viability in soil.
The effect of the expression plasmids on the survivability of the complementary strains differed depending
on the transcriptional regulators. Transcriptional regulators with mostly or partially suppressed effects on
survivability included abgR, ihfA, rpoS, and crp. For these transcriptional regulators, the expression plasmids
compensated for the gene-deficiency effects, confirming their role in long-term survivability in soil. As 50 μM
of IPTG was used for constant induction in the current study, the effects of these transcriptional regulators on
long-term survival in soil could be constant, and they functioned well at certain expression levels. In contrast,
the effects on survivability were not significantly suppressed by expression plasmids when rpoN, ygbI, dps, ompR,
mlc, lrp, cbl, and cadC were deficient, suggesting that the activity phase or expression levels of these transcription
factors were specific to long-term survival in soil. Alternatively, the expression levels of these transcription
factors may have been inappropriate under the induction conditions used in the present study because the
specific IPTG concentrations were used, or the transcription factors expressed by the expression vectors of the
ASKA library were His-tagged, resulting in functional inhibition60. Favorable results were obtained for ΔdinJ
and ΔnhaR and complementary strains. The ΔdinJ strain showed a significant decrease in survival rate compared
to that of the wild-type strain, whereas the DinJ-complemented deficient strain showed an increase in survival
rate. The ΔnhaR strain exhibited increased survivability compared to the wild-type strain, whereas the NhaR-
complemented strain showed a decrease. These effects of DinJ and NhaR suggest that the intensity of their
function is a predominant factor in long-term survivability in soil. Note that, the transcription factors with a

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10- or more-fold reduction in survival at 42 days in two sets of experiments (Exp-1 and Exp-2) were carefully
examined in this study, while eight transcription factors with more than a five-fold reduction included AgaR
(N-acetylgalactosamine repressor), BluR (blue-light sensing regulator), DeoT (DeoR-type regulator), EnvY
(envelope polymeptide regulator), Fur (ferric uptake regulator), PhoB (phosphate regulator), PunR (purine
transporter regulator), and YdeO (not renamed but involved in acid resistance) (Table S1). All of these also
showed significance in the statistical analysis and thus appear to have some influence on long-term survival.
Different soil types have been reported to affect the survival of E. coli61. Although sieved black soil was used in
this study, future experiments with different soil types will verify the general effect.
Understanding the mechanisms of the long-term viability of pathogenic microorganisms in soil and the
natural environment is useful for infectious disease control. The transcription factors affecting long-term E. coli
soil viability identified in the current study were analyzed for conservation among pathogenic microbial species.
Representative pathogenic bacteria from “Soil Borne Human Diseases” published by the European Commission
Joint Research Centre1 and “Global Priority Pathogens List” published by the World Health Organization2,
in which any one of the 14 target transcription regulators is conserved are listed in Table 2. Conservation of
transcription regulators was analyzed using QuickBLASTP (the target percent identity was 50% or more) to
determine high conservation based on amino acid sequences. The results showed that various transcription
factors were conserved in pathogenic bacteria belonging to Proteobacteria, while a few were conserved in those
belonging to Firmicutes, including Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae.
Transcription factors that regulate more than a few dozen genes, such as nucleoid-associated, global, and local
regulators, were highly conserved among bacterial species, unlike sigma factors and single-target regulators,
which were less conserved. This is consistent with the general physiological role of the functions of the identified
transcription factors. Therefore, the effects of the E. coli transcription factors identified in the present study
suggest that the transcription factors themselves and the systems they sense and regulate are of broad importance
among other bacterial species. It should be noted that microbial species with less conserved transcription factors,
as identified in E. coli, likely have similar alternative functions for long-term viability.
The present study was conducted on all transcription factors of a single bacterium and is the first
comprehensive identification of transcription factors required for the long-term survival of bacteria in soil.
The results of this study will be useful in understanding the cues detected by bacteria and the gene functions
that are important for regulating long-term survival in soil. Furthermore, these findings will also be useful for
understanding the physiological functions of soil bacteria and managing bacteria in natural environments. This
includes suppressing soil-borne infections by pathogenic microorganisms and regulating carbon and nitrogen
cycles in the soil, which are related to plant growth and greenhouse gas emissions62,63. Survivability is influenced
not only by transcriptional regulators but also by the genes they control. Transcription factors act both as
activators and repressors. The deficiency of a transcription factor may result in the de-repression or de-activation
of under-regulated genes. Further studies are needed to identify causative genes within the regulon.

Materials and methods

Bacterial strains and plasmids
Escherichia coli BW2511364, its single-gene knockout mutant29, and an expression plasmid from the ASKA clone
library60 were obtained from the E. coli Stock Center (National Bioresource Center, Chiba, Japan). All gene-
deficient strains used are listed in Table S1. Cells were grown as pre-culture in LB medium at 37 °C with constant
shaking at 150 rpm.

Assay for the long-term survivability of E. coli in soil

The flow of the soil survival assay is shown in Fig. 1A. The assay was performed according to the method
described by Somorin et al.18 with some modifications.
The E. coli BW25113 strain (wild-type strain), single-gene knockout mutants, and strains harboring the
expression plasmid were pre-cultured in LB medium for 18 h. When necessary, kanamycin (20 μg mL−1) for
the deficient strains or chloramphenicol (30 μg mL−1) for the strains harboring the expression plasmid was
added to the medium. Next, the culture medium was transferred into a tube and centrifuged (3260 × g, 5 min,
25 ℃) to remove the supernatant. Thereafter, phosphate-buffered saline (PBS [–]) was added to the cell pellets,
resuspended, and centrifuged, and the supernatant was removed twice. After addition to soil, E. coli cells were
suspended in PBS (-) to approximately 2 × 107 cells.
Sieved black soil (VIVAHOME, Saitama, Japan) was used for this assay. The soil was further passed through
a 1-mm sieve to align the particle size of the soil used in the assay. Subsequently, the soil was sterilized using an
autoclave and then carefully dried. The water holding capacity of the adjusted black soil was measured using the
Hilgard method65 and determined to be 1.44 mL g−1. As the water content of the upper layer of soil in the natural
environment averages approximately 60%65, this assay was also conducted by adding E. coli suspended in 864
μL of PBS (−) to dry black soil with a water holding capacity of 1.44 mL g−1, resulting in a water content of 60%.
For this assay, 15-mL plastic tubes were used with their caps loosened during incubation. The air layer was
maintained at 60% humidity and 25 °C using an electronic cold thermal thermohygrostat chamber THR050FB
(Advantec, Tokyo, Japan). One gram of soil per sample was used as one sample. Next, approximately 2 × 107
E. coli cells in 864 μL of PBS (–) were added to the top of the soil in the tube. For plasmid-complemented
transformants, 50 µM IPTG was added to induce transcription factor expression. The test cells were then flash-
centrifuged at 1000 rpm for 10 s to allow penetration throughout the soil.
Viable cells were counted using platelet culture at days 0, 3, 7, 21, and 42 post-incubation. For sampling,
9 mL of PBS (–) was added to the plastic tube, vortexed (2,000 rpm, 20 s) twice, and the supernatant was
collected after 1 min. Subsequently, the supernatant was diluted in multiple steps, spread onto LB agar plates,
and incubated overnight at 37 °C. The number of colonies formed was measured using a Scan300 colony counter

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(Interscience, Saint Nom, France). Three independent biological replicates were prepared for each sampling
point per experimental set, and the number of viable cells in each was determined.

Conservation analysis of transcription factors

All amino acid sequences of the transcription factors analyzed were obtained from PEC, an E. coli database66.
The species of bacteria used for the conservation analysis were based on the lists of microorganisms in “Soil
Borne Human Diseases”1 and “WHO Global Priority Pathogens List”2. Conservation was determined in BLAST
(National Center for Biotechnology Information) using the Quick BLASTP algorithm, which works best if the
target percent identity is ≥ 50% (https://blast.ncbi.nlm.nih.gov/blast/Blast.cgi).

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary
information files. Information on E. coli genes, such as description, the deletion mutant (KO collection) and
expression vector (ASKA library), is based on the profiling of E. coli chromosome database ​(​​​h​t​t​p​s​:/​ ​/​s​h​i​g​e​n​.​ni​ ​g​.​
a​c​.​j​p​/​ec​ ​o​l​i​/​p​e​c​/​​​​)​ ​.​​

Received: 4 October 2024; Accepted: 3 January 2025

References
1. Jeffery, S. & van der Putten, W. H. Soil Borne Human Diseases 24893 (EUR, 2011) (Joint Research Centre—Institute for Environment
and Sustainability, 2011).
2. Tacconelli, E. et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria
and tuberculosis. Lancet Infect. Dis. 18, 318–327 (2018).
3. Lemunier, M. et al. Long-term survival of pathogenic and sanitation indicator bacteria in experimental biowaste composts. Appl.
Environ. Microbiol. 71, 5779–5786 (2005).
4. Cloutier, D. D. & McLellan, S. L. Distribution and differential survival of traditional and alternative indicators of fecal pollution at
freshwater beaches. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.02881-16 (2017).
5. Yao, Z., Zhang, H., Liang, C., Wang, Y. & Wu, Y. Effects of cultivating years on survival of culturable Escherichia coli O157:H7 in
greenhouse soils. J. Food Prot. 82, 226–232 (2019).
6. Sharma, M. et al. Survival of Escherichia coli in manure-amended soils is affected by spatiotemporal, agricultural, and weather
factors in the mid-Atlantic United States. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.02392-18 (2019).
7. Baker, C. A., Lee, S., De, J., Jeong, K. C. & Schneider, K. R. Survival of Escherichia coli O157 in autoclaved and natural sandy soil
mesocosms. PLoS ONE https://doi.org/10.1371/journal.pone.0234562 (2020).
8. Detert, K. & Schmidt, H. Survival of enterohemorrhagic Escherichia coli O104:H4 strain C227/11Φcu in agricultural soils depends
on rpoS and environmental factors. Pathogens https://doi.org/10.3390/pathogens10111443 (2021).
9. Dusek, N., Hewitt, A. J., Schmidt, K. N. & Bergholz, P. W. Landscape-scale factors affecting the prevalence of Escherichia coli in
surface soil include land cover type, edge interactions, and soil pH. Appl. Environ. Microbiol. ​h​t​t​p​s​:​/​/​d​o​i​.​or​ ​g​/​1​0​.​1​1​2​8/​ ​A​E​M​.​0​2​7​1​
4​-​1​7​​​​ (2018).
10. Underthun, K., De, J., Gutierrez, A., Silverberg, R. & Schneider, K. R. Survival of Salmonella and Escherichia coli in two different
soil types at various moisture levels and temperatures. J. Food Prot. 81, 150–157 (2018).
11. Stocker, M. D., Pachepsky, Y. A., Hill, R. L. & Shelton, D. R. Depth-dependent survival of Escherichia coli and enterococci in soil
after manure application and simulated rainfall. Appl. Environ. Microbiol. 81, 4801–4808 (2015).
12. Meyers, B. C. & McLellan, S. L. Influence of nutrients and the native community on E. coli survival in the beach environment. Appl.
Environ. Microbiol. https://doi.org/10.1128/aem.01043-22 (2022).
13. Reed-Jones, N. L., Marine, S. C., Everts, K. L. & Micallef, S. A. Effects of cover crop species and season on population dynamics of
Escherichia coli and Listeria innocua in soil. Appl. Environ. Microbiol. 82, 1767–1777 (2016).
14. Ishihama, A. Functional modulation of Escherichia coli RNA polymerase. Annu. Rev. Microbiol. 54, 499–518 (2000).
15. Shimada, T., Tanaka, K. & Ishihama, A. The whole set of the constitutive promoters recognized by four minor sigma subunits of
Escherichia coli RNA polymerase. PLoS ONE https://doi.org/10.1371/journal.pone.0179181 (2017).
16. van Hoek, A. H., Aarts, H. J., Bouw, E., van Overbeek, W. M. & Franz, E. The role of rpoS in Escherichia coli O157 manure-amended
soil survival and distribution of allelic variations among bovine, food and clinical isolates. FEMS Microbiol. Lett. 338, 18–23
(2013).
17. Ravva, S. V., Cooley, M. B., Sarreal, C. Z. & Mandrell, R. E. Fitness of outbreak and environmental strains of Escherichia coli
O157:H7 in aerosolizable soil and association of clonal variation in stress gene regulation. Pathogens 3, 528–548 (2014).
18. Somorin, Y., Abram, F., Brennan, F. & O’Byrne, C. The general stress response is conserved in long-term soil-persistent strains of
Escherichia coli. Appl. Environ. Microbiol. 82, 4628–4640 (2016).
19. Somorin, Y. et al. Roles for RpoS in survival of Escherichia coli during protozoan predation and in reduced moisture conditions
highlight its importance in soil environments. FEMS Microbiol. Lett. https://doi.org/10.1093/femsle/fnx198 (2017).
20. Ishihama, A., Shimada, T. & Yamazaki, Y. Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of
transcription factors. Nucleic Acids Res. 44, 2058–2074 (2016).
21. Ishihama, A. Building a complete image of genome regulation in the model organism Escherichia coli. J. Gen. Appl. Microbiol. 63,
311–324 (2018).
22. Tierrafría, V. H. et al. RegulonDB 11.0: comprehensive high-throughput datasets on transcriptional regulation in Escherichia coli
K-12. Microb. Genom https://doi.org/10.1099/mgen.0.000833 (2022).
23. Shimada, T., Ogasawara, H. & Ishihama, A. Single-target regulators form a minor group of transcription factors in Escherichia coli
K-12. Nucleic Acids Res. 46, 3921–3936 (2018).
24. Shimada, T., Furuhata, S. & Ishihama, A. Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its
enhancer protein NtrC in Escherichia coli K-12. Microb. Genomics https://doi.org/10.1099/mgen.0.000653 (2021).
25. Shimada, T., Ogasawara, H., Kobayashi, I., Kobayashi, N. & Ishihama, A. Single-target regulators constitute the minority group of
transcription factors in Escherichia coli K-12. Front. Microbiol. https://doi.org/10.3389/fmicb.2021.697803 (2021).
26. Karp, P. D. et al. The EcoCyc database (2023). EcoSal Plus https://doi.org/10.1128/ecosalplus.esp-0002-2023 (2023).
27. Bardsley, C. A. et al. Growth media of Escherichia coli does not affect its survival in soil under static conditions. J. Food Prot. 85,
1842–1847 (2022).
28. Ishihama, A. & Shimada, T. Hierarchy of transcription factor network in Escherichia coli K-12: H-NS-mediated silencing and
Anti-silencing by global regulators. FEMS Microbiol. Rev. 45, fuab032 (2021).

Scientific Reports | (2025) 15:4279 | https://doi.org/10.1038/s41598-025-85609-8 11

www.nature.com/scientificreports/

29. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol.
2(2006), 0008 (2006).
30. Ishihama, A. Adaptation of gene expression in stationary phase bacteria. Curr. Opin. Genet. Dev. 7, 582–588 (1997).
31. Almirón, M., Link, A. J., Furlong, D. & Kolter, R. A novel DNA-binding protein with regulatory and protective roles in starved
Escherichia coli. Genes Dev. 6, 2646–2654 (1992).
32. Azam, T. A. & Ishihama, A. Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition
specificity and DNA binding affinity. J. Biol. Chem. 274, 33105–33113 (1999).
33. Nair, S. & Finkel, S. E. Dps protects cells against multiple stresses during stationary phase. J. Bacteriol. 186, 4192–4198 (2004).
34. Orban, K. & Finkel, S. E. Dps is a universally conserved dual-action DNA-binding and ferritin protein. J. Bacteriol. 204, e0003622
(2022).
35. Reitzer, L. & Schneider, B. L. Metabolic context and possible physiological themes of σ54-dependent genes in Escherichia coli.
Microbiol. Mol. Biol. Rev. 65, 422–444 (2001).
36. Brown, D. R., Barton, G., Pan, Z., Buck, M. & Wigneshweraraj, S. Nitrogen stress response and stringent response are coupled in
Escherichia coli. Nat. Commun. 5, 4115 (2014).
37. Newman, E. B. & Lin, R. Leucine-responsive regulatory protein: a global regulator of gene expression in E. coli. Annu. Rev.
Microbiol. 49, 747–775 (1995).
38. Shimada, T., Saito, N., Maeda, M., Tanaka, K. & Ishihama, A. Expanded roles of leucine-responsive regulatory protein in
transcription regulation of the Escherichia coli genome: genomic SELEX screening of the regulation targets. Microb. Genom. 1,
e000001 (2015).
39. Shimada, T., Takada, H., Yamamoto, K. & Ishihama, A. Expanded roles of two-component response regulator OmpR in Escherichia
coli: genomic SELEX search for novel regulation targets. Genes Cells 20, 915–931 (2015).
40. Fic, E. et al. cAMP receptor protein from Escherichia coli as a model of signal transduction in proteins—a review. J. Mol. Microbiol.
Biotechnol. 17, 1–11 (2009).
41. Grainger, D. C., Hurd, D., Harrison, M., Holdstock, J. & Busby, S. J. Studies of the distribution of Escherichia coli cAMP-receptor
protein and RNA polymerase along the E. coli chromosome. Proc. Natl Acad. Sci. U. S. A. 102, 17693–17698 (2005).
42. Shimada, T., Fujita, N., Yamamoto, K. & Ishihama, A. Novel roles of cAMP receptor protein (CRP) in regulation of transport and
metabolism of carbon sources. PLoS ONE 6, e20081 (2011).
43. Shimada, T., Yoshida, H. & Ishihama, A. Involvement of cyclic AMP receptor protein in regulation of the rmf gene encoding the
ribosome modulation factor in Escherichia coli. J. Bacteriol. 195, 2212–2219 (2013).
44. Kimata, K., Inada, T., Tagami, H. & Aiba, H. A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding
major glucose transporter in Escherichia coli. Mol. Microbiol. 29, 1509–1519 (1998).
45. Plumbridge, J. Regulation of gene expression in the PTS in Escherichia coli: the role and interactions of Mlc. Curr. Opin. Microbiol.
5, 187–193 (2002).
46. Hosono, K., Kakuda, H. & Ichihara, S. Decreasing accumulation of acetate in a rich medium by Escherichia coli on introduction of
genes on a multicopy plasmid. Biosci. Biotechnol. Biochem. 59, 256–261 (1995).
47. Shin, D., Lim, S., Seok, Y. J. & Ryu, S. Heat shock RNA polymerase (E sigma(32)) is involved in the transcription of mlc and crucial
for induction of the Mlc regulon by glucose in Escherichia coli. J. Biol. Chem. 276, 25871–25875 (2001).
48. Carmel, O., Rahav-Manor, O., Dover, N., Shaanan, B. & Padan, E. The Na+-specific interaction between the LysR-type regulator,
NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli. EMBO J. 16, 5922–5929 (1997).
49. Choi, E., Huh, A. & Hwang, J. Novel rRNA transcriptional activity of NhaR revealed by its growth recovery for the bipA-deleted
Escherichia coli at low temperature. Front. Mol. Biosci. 10, 1175889 (2023).
50. Lesniak, J., Barton, W. A. & Nikolov, D. B. Structural and functional features of the Escherichia coli hydroperoxide resistance
protein OsmC. Protein Sci. 12, 2838–2843 (2003).
51. Forst, S., Delgado, J., Ramakrishnan, G. & Inouye, M. Regulation of ompC and ompF expression in Escherichia coli in the absence
of envZ. J. Bacteriol. 170, 5080–5085 (1988).
52. Seo, S. W. et al. Revealing genome-scale transcriptional regulatory landscape of OmpR highlights its expanded regulatory roles
under osmotic stress in Escherichia coli K-12 MG1655. Sci. Rep. 7, 2181 (2017).
53. Ishihama, A. The nucleoid: an overview. EcoSal Plus https://doi.org/10.1128/ecosalplus.2.6 (2009).
54. Hussein, M. J., Green, J. M. & Nichols, B. P. Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by
Escherichia coli. J. Bacteriol. 180, 6260–6268 (1998).
55. Torres, A. G. The cad locus of Enterobacteriaceae: more than just lysine decarboxylation. Anaerobe 15, 1–6 (2009).
56. van der Ploeg, J. R., Eichhorn, E. & Leisinger, T. Sulfonate-sulfur metabolism and its regulation in Escherichia coli. Arch. Microbiol.
176, 1–8 (2001).
57. Prysak, M. H. et al. Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation
through sequence-specific and frame-dependent mRNA cleavage. Mol. Microbiol. 71, 1071–1087 (2009).
58. Hu, Y., Benedik, M. J. & Wood, T. K. Antitoxin DinJ influences the general stress response through transcript stabilizer CspE.
Environ. Microbiol. 14, 669–679 (2012).
59. Gao, Y. et al. Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo. Nucleic Acids
Res. 49, 9696–9710 (2021).
60. Kitagawa, M. et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique
resources for biological research. DNA Res. 12, 291–299 (2005).
61. Alegbeleye, O. et al. Survival behavior of six enterotoxigenic Escherichia coli strains in soil and biochar-amended soils. Environ. Res.
223, 115443 (2023).
62. Itakura, M. et al. Mitigation of nitrous oxide emissions from soils by Bradyrhizobium japonicum inoculation. Nat. Clim. Change 3,
208–212 (2013).
63. Akiyama, H. et al. Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens.
Sci. Rep. 6, 32869 (2016).
64. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc.
Natl Acad. Sci. U. S. A. 97, 6640–6645 (2000).
65. Ohba, H. & Owa, N. Vertical distribution of physico-chemical properties and number of sulfur-oxidizing bacteria in the buried
layer of soil profiles with Marine-reduced sulfur compounds. Soil Sci. Plant Nutr. 51, 379–388 (2005).
66. Yamazaki, Y., Niki, H. & Kato, J. Profiling of Escherichia coli chromosome database. Methods Mol. Biol. 416, 385–389 (2008).

Acknowledgements
We wish to thank the National BioResource Project, National Institute of Genetics, Japan, for providing E. coli
K-12 BW25113, its single-gene deletion mutants, and expression plasmids.

Author contributions
Conceptualization, S.I. and T.S.; methodology, S.N., I.K., K.W., T.K., and T.S.; formal analysis, S.N., I.K., K.W.,
T.K., and T.S.; investigation, S.N., I.K., K.W., T.K., and T.S.; resources, S.I. and T.S.; writing—original draft prepa-

Scientific Reports | (2025) 15:4279 | https://doi.org/10.1038/s41598-025-85609-8 12

www.nature.com/scientificreports/

ration, T.S.; writing—review and editing, S.N., I.K., K.W., T.K., S.I., and T.S.; funding acquisition, S.I. and T.S. All
authors have read and agreed to the published version of the manuscript.

Funding
This research was funded by MEXT Grants-in-Aid for Scientific Research (C) (22K06184) to T.S.

Declarations

Competing interests
The authors declare no competing interests.

Additional information
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