After sampling, screening natural bacteria, and conducting species identification on them, we found that the 2,000 bacterial strains collected belong to 120 genera and a total of 286 species.

The extracted plasmids were verified by agarose nucleic acid electrophoresis, and the results are shown in the figure.

Lane Number	Plasmid Number	Lane Number	Plasmid Number	Lane Number	Plasmid Number
1	pRM 1	14	pRM 14	27	pEM 2
2	pRM 2	15	pRM 15	28	pEM 3
3	pRM 3	16	pRM 16	29	pWM 1
4	pRM 4	17	pRM 17	30	pWM 2
5	pRM 5	18	pRM 18	31	pKM1
6	pRM 6	19	pRM 19	32	pKM2
7	pRM 7	20	pSM 1	33	pKM3
8	pRM 8	21	pSM 2	34	pKM6
9	pRM 9	22	pSM 3	35	pCM1
10	pRM 10	23	pSM 4	36	pCM2
11	pRM 11	24	pSM 5	37	pCM3
12	pRM 12	25	pSM 6	38	pCM4
13	pRM 13	26	pEM 1

Due to time constraints, we only conducted experimental verification on 30 out of the 38 screened natural plasmid-containing bacterial strains.

Salt stress test: The test was carried out under the culture condition with a NaCl concentration of 7%. The growth curves of 30 plasmid-containing bacterial strains in LB medium with 7% NaCl are shown in Figure 1. In the environment containing 7% NaCl, all 30 host bacterial strains grew well, and strain No. 2-8-4 had the highest growth amount.

Alkali stress test: The growth ability in an alkaline environment with pH=10 was tested. In the medium with pH=10, all 30 plasmid-containing bacterial strains could grow normally, and there were differences in the growth amount measured by OD600.

The test results under the alkali stress of pH 10 are shown in Figure 2, and strain No. 2-8-4 had the highest growth amount.

Acid stress test: The test was conducted in an acidic environment with pH=4.9. The growth amounts of the 30 plasmid-containing bacterial strains are shown in Figure 3. Strain No. Y4 had the highest growth amount.

Low-temperature stress test: The test was performed under the culture conditions of 16℃ and 18℃. Under the culture condition of 16℃, 14 bacterial strains including YH4, 2-3-44 and M6-66 grew normally, and their growth curves are shown in Figure 4.

In the low-temperature environment of 16℃, strain Y12 had the highest growth amount. Since 16 plasmid-containing bacterial strains could not grow at 16℃, the test was conducted at 18℃, and the test results at 18℃ are shown in Figure 5.

High-temperature stress test: Initially, a high temperature of 45℃ was set to test the high-temperature resistance of the plasmid-containing bacterial strains. In the test environment of 45℃, the 22 plasmid-containing bacterial strains are as shown in Figure 6, and strain No. 2-8-4 had the highest growth amount.

Verification of Transformation Results of Recombinant Engineering Bacteria

Ten natural plasmid vectors were linearized and then subjected to homologous recombination with the target gene fragment of the Kana⁺ resistance gene.

Among them, 8 natural plasmid vectors and the target gene fragment all amplified a single bright band, while 2 natural plasmids did not amplify any bands.

This indicates that the PCR system is effective, the primers specifically bind to the template, and the products can be used for subsequent homologous recombination.

After the 8 recombinant plasmids were transformed into Escherichia coli BL21 by heat shock, positive clones were all formed on LB plates containing Kana⁺.

The results show that homologous recombination successfully inserted the Ori (Origin of Replication) and Kana⁺ resistance gene into the natural plasmids, and the recombinant plasmids can replicate autonomously and express resistance in the host bacteria.

Results of Stress Tests

The results of the salt tolerance test are shown in Figure 7.

The test results indicate that under the salt condition of 6% NaCl concentration, after plasmid introduction, compared with the control group E. coli BL21, the growth of 7 strains increased except for the engineering bacteria containing the pRM13+K plasmid. The engineering bacteria containing the recombinant plasmid pWM2+K had the highest growth.

It is evident that plasmid pWM2 has a strong alkali tolerance function.

The results of the alkali tolerance test are shown in Figure 8.

The test results indicate that under the alkaline condition of pH=9, after plasmid introduction, compared with the control group E. coli BL21, the growth of all 8 strains increased. Among them, the growth of engineering bacteria containing recombinant plasmids pWM1+K, pRM4+K, and pRM7+K increased most significantly.

The growth of other engineering bacteria containing recombinant plasmids was relatively similar to that of the control group E. coli BL21. It is推测 that plasmids pWM1, pRM4, and pRM7 have strong alkali tolerance functions.

The results of the acid tolerance test are shown in Figure 9.

The test results indicate that under the alkaline condition of pH=4.15, after plasmid introduction, compared with the control group E. coli BL21, the growth of 7 strains increased except for the engineering bacteria containing the pRM13+K plasmid. Among them, the growth of engineering bacteria containing recombinant plasmids pRM4+K and pRM6+K increased most significantly.

It is evident that plasmids pRM4 and pRM6 have strong acid tolerance functions.

The results of the low-temperature tolerance test are shown in Figure 10.

The test results indicate that under the low-temperature condition of 18℃, after plasmid introduction, compared with the control group E. coli BL21, the growth of all 8 engineering strains was lower than that of the control.

It is speculated that none of the 8 plasmids used in this study have low-temperature tolerance ability.

The results of the high-temperature tolerance test are shown in Figure 11.

The test results indicate that under the high-temperature condition of 45℃, after plasmid introduction, compared with the control group (E. coli BL21), the growth of 7 out of the engineering bacteria containing recombinant plasmids increased, except for those harboring the pWM1+K and pRM+K plasmids.

Among them, the engineering bacterium containing the recombinant plasmid pRM4+K showed the most significant increase in growth.

It is speculated that plasmid pRM4 has a strong high-temperature tolerance function.

ORF Prediction Results

The functions of proteins encoded by plasmid sequences that appear multiple times in different plasmids mainly fall into four categories:

The first category is involved in the replication and repair of plasmid DNA, such as sequences encoding replication initiation proteins, plasmid recombination enzymes, DNA repair and recombination proteins, and tRNA ligases.

The second category includes sequences that participate in encoding the host's physiological metabolic activities and energy generation and metabolism. These sequences have functions of encoding triosephosphate isomerases, regulator aspartate phosphatase E, alpha-1,3-glucan synthases, and phosphoglycolate phosphatases.

The third category of encoded proteins mainly functions in substance transport and signal transduction. Examples include sequences encoding maltose/maltodextrin transport system permease protein MalF, proteins of the NRT1/PTR family, and phosphate import ATP-binding proteins.

The fourth category consists of ORFs (Open Reading Frames) that encode proteins with specific functions, such as probable F-box proteins and small heat shock protein C4. F-box proteins are important components involved in the substrate ubiquitination process and participate in the assembly of ubiquitin ligases. In contrast, small heat shock protein C4 is induced to express when cells are exposed to various stresses (such as high temperature, oxidative stress, etc.) and plays a role in protecting cells from stress damage.

From the basic physiological metabolism of bacteria to stress responses, ORFs with different functions are predicted to be involved in these processes. The following are some predicted results of plasmid ORFs.

Plasmid	Strain	ORF	Coding Protein	No Significant Similarity
pRM 4	2-8-4	ORF9	Putative replication protein	7
		ORF8	Kanamycin nucleotidyltransferase
		ORF11	Flagellar brake protein YcgR
		ORF5	Probable F-box protein
		ORF6	DNA replication and repair protein
pRM 6	FH 2-3-8	ORF9	Protein NRT1/ PTR FAMILY	11
		ORF12	RNA polymerase II degradation factor 1
		ORF10	Plasmid recombination enzyme
		ORF1	Maltose/maltodextrin transport system permease protein MalF
pRM 7	M6-66	ORF6	DNA primase TraC	3
		ORF1	Plasmid recombination enzyme type 3
		ORF5	Major capsid protein
pRM 8	M6-69	ORF3	Methyl-accepting chemotaxis protein III	3
		ORF4	Mobilization protein MobL
		ORF1	Type II methyltransferase M.AgeI
		ORF2	Chromosome partitioning protein ParA
		ORF8	Tyrosine recombinase XerD (+ 6310-6876)
		ORF9	Transposase InsF for insertion sequence IS3A (+ 346-693)
pRM 13	2-3-44	ORF9	Response regulator aspartate phosphatase E	5
		ORF4	Nipped-B-like protein B
		ORF1	Netrin receptor DCC
		ORF3	Replication initiation protein
pWM 1	3-6-1	ORF4	Replication initiation protein	17
		ORF11	DNA replication and repair protein
		ORF5	Putative O-methyltransferase MUL_4520
		ORF16	Endoglucanase 16
		ORF9	Triosephosphate isomerase
pWM 2	YH4	ORF17	Triosephosphate isomerase	19
		ORF12	Restriction of telomere capping protein 5
		ORF19	Transcription antitermination protein NusB
		ORF13	Putative O-methyltransferase MUL_4520
		ORF3	Endoglucanase 16
pSM 4	YM-5	ORF11	Replication initiation protein	18
		ORF7	Transposon Tn552
		ORF3	Plasmid-partitioning protein ParA
		ORF20	Putative toxin HigB3
		ORF23	Antitoxin HigA1

SDS-PAGE analysis and double enzyme digestion verification Result

Based on the frequency of ORF occurrence and their functions, we selected 12 ORFs.

After SDS-PAGE analysis and Restriction Enzyme Verification, we constructed recombinant engineering bacteria.

We conducted stress test cultivation under three conditions: pH=4.15, pH=9, and 6% NaCl.

The protein gel images are shown in Figures a, b, and c.

Figure	Lane	Protein
Figure a	Lane 1	Presumptive replication protein, uninduced
	Lane 2	Presumptive replication protein, induced
	Lane 3	Chromosome partitioning protein, uninduced
	Lane 4	Chromosome partitioning protein, induced
	Lane 5	Main partitioning protein parA, uninduced
	Lane 6	Main partitioning protein parA, induced
	Lane 7	Induced head protein, uninduced
	Lane 8	Induced head protein, induced
Figure b	Lane 1	Small heat shock protein, uninduced
	Lane 2	Small heat shock protein, induced
	Lane 3	Replication initiation protein, uninduced
	Lane 4	Replication initiation protein, induced
	Lane 5	Induced Rip (plasmid replication initiation protein), uninduced
	Lane 6	Induced Rip (plasmid replication initiation protein), induced
	Lane 7	Induced Rip (plasmid replication initiation protein), uninduced
	Lane 8	Induced Rip (plasmid replication initiation protein), induced
Figure c	Lane 1	DNA replication and repair protein, uninduced
	Lane 2	DNA replication and repair protein, induced
	Lane 3	Triose phosphate isomerase, uninduced
	Lane 4	Triose phosphate isomerase, induced
	Lane 5	Small heat shock protein, uninduced
	Lane 6	Endoglucanase, induced
	Lane 7	Endoglucanase 16, uninduced
	Lane 8	Endoglucanase 16, induced

The results of double enzyme digestion verification are shown in the figure.

Results of Stress Tests

After stress tests cultivation under pH=4.15, pH=9, and 6% NaCl conditions, we have obtained the following new findings:

1. ORF3 and ORF7 derived from pSM4 both exhibit tolerance under 6% NaCl, pH=4.15 and pH=9 environments, as shown in Figure 12 and Figure 13 respectively.
2. ORF5 derived from pRM18 exhibits tolerance under 6% NaCl and pH=9 environments, as shown in Figure 13 and Figure 14 respectively.
3. ORF10 derived from pRM17 exhibits tolerance under pH=4.15 and pH=9 environments, as shown in Figure 13 and Figure 14 respectively.

In contrast, ORF2, ORF4, ORF6, ORF8, ORF9, ORF11 and ORF12 all exhibit good tolerance under the alkaline condition of pH=9. ORF1 exhibits good tolerance under the acidic condition of pH=4.15.