Liaoqin Zhang1
Yong Xu- 1School of Life Science, Anhui Agricultural University, Hefei, China
- 2Institute of Clinical Medicine, Anhui Academy of Medical Sciences, Hefei, China
Klebsiella pneumoniae is a leading cause of healthcare-associated infections, with emerging strains exhibiting both multidrug resistance and hypervirulence, largely mediated by plasmid-encoded genes. Poly (A) polymerase I, encoded by pcnB, plays a key role in RNA degradation and plasmid copy number control, yet its global regulatory impact in K. pneumoniae remains unclear. Here, we constructed a pcnB deletion mutant in K. pneumoniae ATCC 13883 using CRISPR-Cas9 and examined its effects on chromosomal virulence factors and plasmid-borne resistance. Deletion of pcnB impaired bacterial growth and metabolic activity, reduced biofilm formation, but unexpectedly enhanced siderophore and exopolysaccharide production via upregulation of chromosomal virulence genes. In contrast, pcnB deletion drastically reduced the copy number and stability of a ColE1-type plasmid carrying a spectinomycin resistance gene (aadA), leading to decreased aadA expression and a twofold reduction in antibiotic resistance. These findings reveal the dual role of pcnB as a repressor of chromosomal virulence genes and an activator of plasmid maintenance, highlighting its potential as a novel target for anti-virulence and anti-resistance strategies.
1 Introduction
Klebsiella pneumoniae (KP) is a Gram-negative opportunistic pathogen belonging to the Enterobacteriaceae family. As a common gut commensal, it ranks as the second most prevalent opportunistic pathogen in clinical settings (Assoni et al., 2021). This bacterium can cause various severe infections including pneumonia, meningitis, liver abscesses, and bloodstream infections (Breurec et al., 2016). In recent years, the widespread use of antibiotics has led to the emergence of KP strains exhibiting both multidrug resistance and hypervirulence (Holt et al., 2015). These KP strains result in significantly increased morbidity and mortality rates, and thereby impose considerable challenges on clinical treatment.
The pathogenicity of KP is attributed to multiple virulence factors, including capsular polysaccharides, siderophores, flagella, and resistance determinants such as carbapenemases (Chen et al., 2024). The expression levels of these factors directly influence the pathogenic capability of this bacterial species. Similar to Escherichia coli (E. coli), KP possesses a highly plastic genome capable of acquiring various resistance and virulence genes through horizontal gene transfer (Wyres et al., 2019). This characteristic facilitates the enhancement of virulence and development of antibiotic resistance, rendering conventional antibiotic therapies increasingly ineffective (Liu et al., 2021). Elucidating the transmission mechanisms of resistance and virulence genes, as well as deciphering the co-evolutionary relationship between bacteria and plasmids, will provide crucial theoretical basis for developing novel anti-bacterial strategies.
To address the dual challenges of multidrug resistance and hypervirulence in KP, significant efforts have been devoted to developing inhibitors targeting various pathogenic mechanisms, such as biofilm formation, drug efflux pumps, and β-lactamases (Lomovskaya et al., 2017; Mahrous et al., 2023; Vieira Da Cruz et al., 2023). Additionally, genetically disrupting key virulence mechanisms, including siderophore biosynthesis, capsule synthesis, and biofilm formation, has shown promising antibacterial effects (Han et al., 2022; Bina et al., 2023). However, current antimicrobial strategies often lack broad-spectrum efficacy, as most inhibitors target individual resistance or virulence genes, leaving other pathogenic mechanisms unaffected. In response to this limitation, ColE1-type plasmids have emerged as a new focus in the search for antibacterial targets, since these extrachromosomal elements are frequently identified as carriers of both resistance and virulence genes (Ares-Arroyo et al., 2021). Furthermore, experimental data have indicated that the presence of high-copy-number plasmids in KP is closely associated with multidrug resistance and hypervirulence phenotypes, playing a key role in its evolutionary trajectory (Ramirez et al., 2019). Therefore, targeting the mechanisms that regulate plasmid copy number could simultaneously disrupt the maintenance of all plasmid-borne resistance and virulence genes.
Based on current knowledge, the replication of ColE1-type plasmids is primarily regulated by the antisense RNA system involving RNA I and RNA II, while their copy number and stability are further modulated by poly (A) polymerase I (PAP I) (Cesareni et al., 1991). PAP I, encoded by the pcnB gene, was initially identified in E. coli for its ability to catalyze the addition of adenosine monophosphate (AMP) residues to the 3′ end of RNA, forming a poly (A) tail that decreases RNA stability and promotes its degradation (Lopilato et al., 1986; Jones, 2021). Subsequent studies revealed that PAP I plays a crucial role in controlling the copy number of ColE1-type plasmids in E. coli (Sarkar et al., 2002). Deletion of pcnB in E. coli leads to a sharp decrease in the copy number of endogenous ColE1-type plasmids (Wellner et al., 2025). Current evidence indicates that PAP I-mediated polyadenylation, encoded by pcnB, promotes the degradation of small RNAs that inhibit plasmid replication. Consequently, deletion of pcnB leads to the accumulation of these inhibitory sRNAs, resulting in decreased plasmid copy number and thereby reduced expression of plasmid-encoded genes (Schubert et al., 2025). However, the net impact of these opposing regulatory trends on plasmid gene expression remains unclear. Given that the high virulence and drug resistance of KP are primarily attributed to plasmid-encoded genes (Shankar et al., 2020), elucidating the regulatory role of pcnB in plasmid gene expression will provide important insights into the molecular mechanisms underlying KP virulence and drug resistance, and establish a theoretical basis for evaluating pcnB as a potential target for inhibiting bacterial virulence and resistance.
To address this, we selected the wild-type K. pneumoniae ATCC 13883 strain (Kp13883), which exhibits low virulence and high transformation efficiency, as our experimental model. By introducing an exogenous plasmid carrying a spectinomycin resistance gene and subsequently generating a pcnB knockout mutant using CRISPR-Cas9 technology, we systematically examined the differential regulatory effects of pcnB deletion on both chromosomally encoded virulence factors and plasmid-mediated drug resistance. This study aims to uncover the distinct regulatory mechanisms of pcnB on chromosomal and plasmid gene expression, thereby exploring its potential as a novel target for anti-infective drug development.
2 Materials and methods
2.1 Plasmids, bacterial strains, and growth conditions
The wild-type Kp13883 served as the parental strain in this study. All strains and plasmids used are listed in Table S1. The wild-type strain, its pncB deletion mutant, and the complementation strain were designated as WT, ΔpcnB and ΔpcnB (pBBR-pcnB), respectively. Strains harboring the ColEI-type plasmid pEcgRNA (spectinomycin resistant) were denoted as WT- pEcgRNA and ΔpcnB-pEcgRNA. Bacterial cultures were routinely grown in Luria-Bertani (LB) medium and M9 minimal medium (Table S2) at 37 °C with 180 rpm shaking or on LB agar plates overnight. For strains carrying temperature-sensitive plasmids, a 42 °C incubation was applied to facilitate plasmid curing. When required, antibiotics were supplemented at the following concentrations: 50 μg/mL kanamycin (Kan), spectinomycin (Spec), and apramycin (Apr).
2.2 Preparation of competent cells and transformation
Competent cells were prepared using an optimized arabinose induction protocol. Briefly, Kp13883 overnight culture was diluted 1:50 in fresh LB medium and incubated at 37 °C with shaking until reaching OD600 of 0.2–0.3. L-arabinose was then added to a final concentration of 0.1% (w/v) to induce competence-related genes. Upon reaching OD600 of 0.5, cells were harvested by centrifugation at 4,000 × g for 10 min at 4 °C and washed four times with ice-cold 10% glycerol. Finally, the cell pellet was resuspended in a minimal volume of 10% glycerol and either used immediately or stored at −80 °C (Jiang et al., 2016; McConville et al., 2021).
For electroporation, 1 μg plasmid DNA was mixed with 100 μL competent cells on ice. The mixture was transferred to a pre-chilled 2-mm electroporation cuvette and pulsed at 2.5 kV using a Gene Pulser system (Bio-Rad). Immediately after electroporation, 900 μL SOC recovery medium was added, and cells were incubated at 37 °C for 1 h with shaking. Aliquots (10 μL and 50 μL) were plated on LB agar containing appropriate antibiotics and incubated overnight at 37 °C.
2.3 Plasmid construction of mutagenesis
All PCR primers were designed using SnapGene software (v5.2) based on the Kp13883 genomic sequence (NCBI accession no. CP009208) and are listed in Table S1. The pcnB deletion mutant was generated using pCasKP-apr/pSGKP-km mediated CRISPR-Cas9 editing system as previously described (Sun et al., 2019), with modifications. Specifically: (i) Two ~ 600-bp homology arms flanking the pcnB gene were amplified as a single fragment and cloned into pSGKP-km. (ii) Guide RNA (gRNA) targeting pcnB was designed using CHOPCHOP online tool (ref) (Labun et al., 2019) and cloned into pSGKP-km plasmid. (iii) The constructed plasmids were sequentially transformed into Kp13883 harboring pSGKP-km through electroporation. (iv) Positive clones were selected on LB plates containing triple antibiotics (Kan and Apr) and verified by colony PCR using pcnB-specific primers (P1/P2 in Table S1) and sequencing.
2.4 Supplementation of pcnB mutants
To confirm that the observed phenotype was caused by the pcnB gene deletion. The coding sequence of the pcnB gene was first amplified fromWT strain genomic DNA using specific primers (Table S1). These primers were designed with 20-bp homologous arms at their 5′ ends complementary to the termini of the pBBR1MCS-2 vector linearized by inverse PCR. The purified pcnB PCR fragment was then inserted into the multiple cloning site (MCS) of the linearized vector, with pcnB gene expression driven by the vector’s native lac promoter and ribosome binding site (RBS). Assembly was performed using the Ultra-Universal One Step Seamless Cloning Mix kit (CWBIO, China) at 50 °C for 15 min. The assembled product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing kanamycin for selection. Finally, positive clones were verified by colony PCR. The plasmid was then extracted and electroporated into the ΔpcnB strain to construct the complementing strain ΔpcnB (pBBR-pcnB), which was finally validated by bacterial suspension PCR.
2.5 Bacterial growth curve analysis
WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains were cultured overnight in LB medium at 37 °C with 180 rpm shaking. Three biological replicates per strain were diluted to an OD600 of 0.1 in fresh LB medium. Growth was monitored by measuring OD600 values at 1-h intervals for 12 h under identical culture conditions, followed by growth curve construction.
2.6 Biofilm formation assay and viability testing
Overnight cultures in LB medium were adjusted to 0.5 McFarland standard. Aliquots (10 μL) of bacterial suspension plus 190 μL fresh LB medium were added per well in 96-well plates (n = 6 biological replicates). After 36-h incubation at 37 °C, planktonic cells were removed and wells were washed twice with PBS. Biofilms were fixed with 200 μL 4% paraformaldehyde for 30 min, air-dried, then stained with 0.1% crystal violet (200 μL, 20 min). Excess stain was removed by PBS washing, and bound dye was solubilized with 200 μL 33% glacial acetic acid for spectrophotometric measurement at 570 nm (LB medium as negative control).
Bacterial viability was assessed using fluorescein diacetate (FDA) which serves as a substrate for detecting metabolically active bacteria. Its hydrolysis by intracellular esterases generates fluorescent products, allowing quantitative measurement of hydrolytic enzyme activity in bacterial biofilms and subsequent evaluation of metabolic vitality. A 10 mg/mL FDA stock solution (in organic solvent) was prepared for light-protected storage. Working solution was prepared fresh in PBS with vortex mixing. Cultures at logarithmic and stationary phases were OD600-normalized and tested in triplicate. Reaction mixtures (50 μL total) containing 45 μL FDA working solution and 5 μL bacterial suspension were incubated at 37 °C for 20 min before fluorescence spectrophotometry. The excitation and emission wavelengths for FDA detection are 490 nm and 520 nm, respectively.
2.7 Siderophore quantification
Siderophore production was assessed using the Chrome Azurol S (CAS) assay (Rathod et al., 2024). The CAS solution was prepared by combining 2 mM CAS, 1 mM FeCl3·6H2O (in 10 mM HCl), and piperazine buffer (pH 5.6) with Hexadecyltrimethylammonium bromide (HDTMA) in a 100-mL cylinder. A 0.2 M sulfosalicylic acid shuttle solution was used as the iron chelator. Bacterial cultures were grown to log or stationary phase in low-phosphate minimal medium buffered with HEPES (to avoid phosphate interference). For detection, 0.5 mL CAS solution was mixed with 0.5 mL culture supernatant, followed by 10 μL shuttle solution. After 5 min incubation, absorbance at 630 nm was measured against appropriate controls. Siderophore units were calculated as [(An-As)/An] × 100, where An and Aₛ represent reference and sample absorbances, respectively.
2.8 Extracellular polysaccharide (EPS) measurement
EPS was quantified by anthrone-sulfuric acid method (Jing et al., 2022). Overnight cultures (7 mL) were centrifuged (5,000 × g, 15 min), washed with PBS, then treated with 0.1 M NaOH (2 h, room temperature). After centrifugation (3,500 × g, 15 min, 4 °C), supernatants were mixed with 0.2% anthrone-sulfuric reagent (1:3 ratio; 0.2 g anthrone in 100 mL concentrated H2SO4), heated at 100 °C for 7 min, and measured at 630 nm after cooling.
2.9 Plasmid copy number determination
Log-phase bacterial cultures were harvested after OD600 normalization, and total DNA was extracted for relative quantification. Plasmid copy numbers (PCN) were calculated by qPCR using a chromosome-encoded reference gene gapA for normalization (Hao et al., 2025). To specifically target the plasmid, we designed a pair of qPCR primers (Table S1) amplifying a 150-bp fragment within the SpecR (aadA gene encoding an aminoglycoside adenyltransferase) gene of pEcgRNA. and relative copy number was calculated using the comparative threshold cycle 2−ΔΔCt method.
2.10 Minimum inhibitory concentration (MIC) testing
The optimized broth microdilution method was used to determine the MICs of Spec against the WT, ΔpcnB, WT-pEcgRNA, and ΔpcnB-pEcgRNA strains. The assay adhered to the fundamental framework of the Clinical and Laboratory Standards Institute (CLSI) guidelines, with a refinement step incorporated to enhance accuracy. Initially, a standard two-fold serial dilution was performed for preliminary screening to determine the approximate MIC range (e.g., testing concentrations of 256, 128, 64, 32, and 16 μg/mL). Subsequently, a more concentrated gradient dilution was conducted within the critically identified range to precisely determine the MIC value. During the experiment, within the range of 64 μg/mL to 16 μg/mL, a series of intermediate concentrations (e.g., 56, 48, 40, 32, and 24 μg/mL) were additionally tested. Three technical replicates and blank controls were included per condition. The MIC was defined as the lowest antibiotic concentration preventing visible growth after incubation.
2.11 RNA quantification and gene expression analysis
Total RNA was extracted using Bacterial RNA Kit (Tiangen, China) and reverse-transcribed with FastKing RT Kit (Tiangen, China) following manufacturer’s protocols. Quantitative PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, USA) using 16S rRNA as endogenous control. Relative gene expression was detected using specific primers (Table S1) and calculated via 2−ΔΔCt method.
3 Results
3.1 Construction of ΔpcnB mutant, off-target detection and growth analysis
The pcnB gene in Kp13883 was precisely deleted using the CRISPR-Cas9 editing system. Colony PCR screening followed by sequencing confirmed the successful generation of mutant strain (ΔpcnB) with complete open reading frame deletion. In addition, we also constructed the pcnB-complemented strain (Figure S1).
To validate the specificity of the target sequence, potential off-target sites were identified using CAS-OFFinder (allowing up to 4 mismatches). PCR amplification was then performed using genomic DNA from the ΔpcnB strain. The successful amplification of correctly sized products (~1,200 bp) from all predicted off-target sites indicates the absence of substantial off-target editing at these loci (Figure S1; Table S3).
To investigate the physiological impact of pcnB deletion, we compared growth kinetics WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains in nutrient-rich LB and minimal M9 media. Starting from standardized inocula (initial OD600 ≈ 0.1), culture densities were monitored spectrophotometrically at 1-h intervals for 12 h. Growth curve analysis revealed that in LB medium, although the ΔpcnB mutant entered log-phase normally, its maximum specific growth rate (μ_max) was 13.6% lower than WT (p < 0.01), while theΔpcnB (pBBR-pcnB) strain showed a significant but smaller reduction of 7.8% compared to the ΔpcnB (p < 0.05) (Figure 1A). Notably, three strains reached stationary phase after 4 h with comparable final OD600 absorption (p > 0.05) indicating pcnB deletion does not affect ultimate biomass accumulation.
Figure 1. Growth curves of the WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains in LB (A) and M9 medium (B). The black curve represents the WT strain, the red curve represents the ΔpcnB strain, and the blue curve represents the complement strain ΔpcnB (pBBR-pcnB). The bacteria were shaken at 37 °C (180 rpm), and the OD600 value was measured every hour to draw the growth curve. The WT and ΔpcnB strains were compared using a two-tailed Student’s t-test at the 1, 2, 3, 4, and 5 h time points. The error bars represent the SEM (n = 3, **, p < 0.01 relative to WT).
More pronounced growth defects were observed in M9 minimal medium (Figure 1B). Compared to the WT, the ΔpcnB strain exhibited a 22.3% reduction in μ_max (p < 0.001) with prolonged logarithmic phase duration, and the ΔpcnB (pBBR-pcnB) strain exhibited a 5.7% reduction. This growth retardation suggests impaired metabolic efficiency under nutrient-limiting conditions, but the ΔpcnB (pBBR-pcnB) was able to partially restore this impairment. However, the growth defect of the ΔpcnB strain was compensated upon entering stationary phase, with no significant differences in growth parameters observed at other time points, compared with the WT strain.
3.2 Modulation of pcnB in cellular viability and biofilm formation in KP
To further elucidate the physiological role of pcnB, we assessed metabolic activity using fluorescein diacetate (FDA) hydrolysis assays. Synchronized measurements of OD600 and FDA fluorescence (excitation/emission: 490/520 nm) were carried out. To account for differences in cell density, FDA fluorescence intensities were normalized to the corresponding OD600 values. As shown in Figure 2A, the WT strain exhibited significantly higher metabolic activity than ΔpcnB in LB medium, and importantly, the ΔpcnB (pBBR-pcnB) strain partially restored this activity in the ΔpcnB. During the logarithmic phase, the fluorescence intensity of WT (137,330 ± 4,856 a.u.) was 12.6% higher than that of ΔpcnB (119,944 ± 4,274 a.u.; p < 0.01), and 5.4% higher than that of the ΔpcnB (pBBR-pcnB) strain (129,809 ± 3,976 a.u.; p > 0.05). This disparity further increased in the stationary phase, where WT reached 186,392 ± 4,765 a.u. compared to 158,509 ± 5,034 a.u. for ΔpcnB (p < 0.001), representing a 15% reduction in metabolic activity upon pcnB deletion, while ΔpcnB (pBBR-pcnB) strain (172,742 ± 5,342 a.u; p < 0.05) partially restored the activity, demonstrating a gene dosage effect.
Figure 2. Bacterial vitality and biofilm formation ability of WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains. (A) Comparison of bacterial vitality in WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains at both the logarithmic and stationary growth phases. (B) Biofilm formation ability of WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains. Data points represent the mean of three biological replicates, each of which was averaged from three technical replicates. The error bars represent the SEM. Statistics: two-way ANOVA (A) and one-way ANOVA (B). ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Given that biofilm formation is directly influenced by bacterial viability and plays a crucial role in antibiotic resistance, we next investigated whether the observed reduction in cell viability affected biofilm development. To investigate the effect of pcnB on biofilm formation in KP, we quantified the biofilm biomass of WT, ΔpcnB and ΔpcnB (pBBR-pcnB) strains using crystal violet staining. OD570 measurements revealed that after 36 h of cultivation in LB medium, the biofilm OD570 value of WT was 0.364 ± 0.02, In contrast, the value forΔpcnB (pBBR-pcnB) was reduced to 0.298 ± 0.02 (p < 0.05), while that of ΔpcnB was significantly reduced to 0.181 ± 0.01 (p < 0.001) (Figure 2B). Although three strains exhibited relatively weak biofilm-forming capacity in LB medium (OD570 < 0.4), pcnB deletion still resulted in a 50% reduction in biofilm formation, demonstrating that pcnB positively regulates biofilm development in KP.
3.3 Effect of pcnB deletion on chromosomal virulence gene expression in KP
To elucidate the regulatory role of pcnB deletion in chromosomally encoded virulence factors of KP, we quantified the production of siderophores and EPS in WT, ΔpcnB (pBBR-pcnB) and ΔpcnB strains.
The chrome azurol S (CAS) assay revealed that during logarithmic growth, the iron-chelating capacity of ΔpcnB showed significant difference compared to WT (p < 0.05), while the ΔpcnB (pBBR-pcnB) strain showed a partial restoration (p > 0.05; Figure 3A; Figure S3). However, upon entering stationary phase, ΔpcnB exhibited a 5.4% increase (p < 0.01) in siderophore units relative to WT. Moreover the siderophore level in ΔpcnB was also 3.7% higher than that of the ΔpcnB (pBBR-pcnB) strain (p < 0.05). indicating enhanced iron acquisition capability upon pcnB deletion during late growth phase. Anthrone-sulfuric acid assay for EPS quantification demonstrated that after OD600 normalization, the insoluble EPS yield in ΔpcnB (0.367 ± 0.01) was 0.1-fold higher than WT (0.331 ± 0.01). EPS production in the ΔpcnB (pBBR-pcnB) strain was somewhat lower than that in the ΔpcnB strain (0.342 ± 0.01; Figure 3B).
Figure 3. Changes in bacterial virulence factor and related gene expression. (A) Comparison of bacterial siderophore content in WT, ΔpcnB, and ΔpcnB (pBBR-pcnB) strains at both the logarithmic and stationary growth phases. (B) Comparison of bacterial EPS content in WT, ΔpcnB, and ΔpcnB (pBBR-pcnB) strains. (C) Relative mRNA levels of entD, fepD, and iroE in the ΔpcnB strain versus WT strain. (D) Relative mRNA levels of rfbA, relA, and wbbM in ΔpcnB strain versus WT. Data points represent the mean of three biological replicates, each of which was averaged from three technical replicates. The error bars represent the SEM. Statistics: two-way ANOVA (A), one-way ANOVA (B) and t-test (C,D). ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Quantitative PCR analysis showed upregulation of siderophore biosynthesis genes entD, fepD, and iroE in ΔpcnB by 1.5-fold, 1.8-fold, and 4-fold, respectively, (Figure 3C). More pronounced upregulation was observed in EPS synthesis genes rfbA, relA, and wbbM, with 18-fold, 3-fold, and 8-fold increases, respectively, (Figure 3D). These findings demonstrate that pcnB deletion enhances KP’s siderophore and EPS production through transcriptional activation of virulence-associated genes.
3.4 Effect of pcnB deletion on spectinomycin resistance of KP mediated by pEcgRNA
Considering the potential for intrinsic spectinomycin resistance on the KP chromosome, we sought to rule out any genome background-associated increase in basal resistance post-knockout. We therefore measured the spectinomycin MIC for WT and ΔpcnB. Table 1 shows that without pEcgRNA, the MIC of WT was 48 μg/mL, significantly greater than the 32 μg/mL of ΔpcnB, suggesting that pcnB deficiency also compromises KP’s intrinsic antibiotic tolerance.
Table 1. MICs for WT and ΔpcnB.
To investigate the role of pcnB in regulating exogenous resistance plasmids, we introduced the ColE1-type plasmid pEcgRNA (carrying the aadA gene encoding an aminoglycoside adenylyltransferase) into WT and ΔpcnB strains (designated WT-pEcgRNA and ΔpcnB-pEcgRNA). Broth microdilution assays revealed that the Spec MIC of WT-pEcgRNA was 11 mg/mL, whereas ΔpcnB-pEcgRNA exhibited a 2-fold reduction in MIC (Table 2), In summary, pcnB knockout concurrently reduced both chromosomal basal tolerance and plasmid-mediated acquired resistance. However, the difference between the respective decreases (12 μg/mL vs. 11 mg/mL) is negligible, indicating compromised spc resistance upon pcnB deletion.
Table 2. MICs for WT-pEcgRNA and ΔpcnB-pEcgRNA.
Quantitative PCR (qPCR) analysis of plasmid copy number (PCN), using the single-copy chromosomal gene gapA for normalization, demonstrated an 80% decrease in PCN for ΔpcnB-pEcgRNA compared to WT-pEcgRNA (Figure 4A). Additionally, transcriptional analysis of the aadA gene (normalized to 16S rRNA) showed a 45% reduction in expression in ΔpcnB-pEcgRNA (Figure 4B). These results suggest that pcnB deletion diminishes Spec resistance by reducing both ColE1 plasmid stability and resistance gene expression.
Figure 4. Relative plasmid copy number and expression of plasmid-encoded genes in WT versus ΔpcnB strains. (A) Relative plasmid copy number of pECgRNA in ΔpcnB strain versus WT strain. The copy number was determined by qPCR using the chromosomal gapA gene for normalization and is presented relative to the WT strain, which was set to 100%. (B) Relative gene expression of aadA in ΔpcnB strain versus WT strain. Data points represent the mean of three biological replicates, each of which was averaged from three technical replicates. The error bars represent the SEM. Statistics: t-test. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
4 Discussion
In recent years, the increasing prevalence of hypervirulent and multidrug-resistant KP, particularly the emergence of carbapenem-resistant hypervirulent KP (CR-hvKP), has posed significant challenges to clinical anti-infective therapy (Turton et al., 2019). The pathogenicity of these strains is commonly mediated by plasmid-carried virulence and resistance genes, making the study of key host factors regulating plasmid stability and gene expression crucial for developing novel antibacterial strategies. This study focused on the pcnB gene, which encodes PAP I that participates in both RNA degradation regulation and direct control of ColE1-type plasmid copy number. Using CRISPR-Cas9 technology, we successfully constructed a pcnB deletion mutant and systematically evaluated its dual functions in regulating chromosomal virulence factor expression and exogenous plasmid-mediated resistance. Moreover, the subsequent construction of ΔpcnB (pBBR-pcnB) strain, which partially restored the key phenotypic defects, strongly supports the conclusion that pcnB plays a central role in coordinating these physiological processes.
Building on the previously reported role of pcnB (PAP I) in mRNA degradation, our results demonstrated (Figures 1A,B) that pcnB deletion significantly affected bacterial physiological states. In terms of growth characteristics, the ΔpcnB strain showed markedly reduced maximum growth rate during the logarithmic phase, with more pronounced delay in minimal M9 medium. This phenomenon suggests that pcnB deletion may impair metabolic efficiency. Notably, although logarithmic growth retardation, the stationary phase biomass remained unchanged, it exhibits a growth lag rather than a permanent growth defect, which is consistent with the observation by Francis & Laishram that the ΔpcnB strain displays a dynamic adaptive capacity due to increased mRNA stability under nutrient-restricted conditions (Francis and Laishram, 2021). Furthermore, normal metabolic activity may be sustained through compensatory mechanisms mediated by other RNA-metabolizing enzymes (such as RNase R or PNPase) (Gerdes et al., 2003; Babina et al., 2023). This growth delay likely results from gene expression reprogramming caused by altered RNA stability.
Regarding virulence and plasmid-mediated stress tolerance regulation, pcnB deletion exhibited a remarkable “double-edged sword” effect. On one hand, siderophore and EPS production significantly increased during stationary phase (Figures 3A,B), accompanied by elevated transcription levels of related genes (e.g., entD, fepD, iroE, rfbA, relA, wbbM) (Figures 3C,D). These data suggest that pcnB may indirectly suppress virulence factor expression by promoting degradation of these virulence-associated mRNAs. By promoting the polyadenylation of structured RNAs, pcnB ensures their efficient turnover; its absence leads to the accumulation of these transcripts, resulting in genome-wide expression dysregulation and pleiotropic effects on bacterial physiology (Maes et al., 2017). These results provide the first molecular evidence that pcnB serves as a repressive regulator in the KP chromosomal virulence network. On the other hand, pcnB deletion caused an 80% reduction in ColE1-type plasmid copy number (Figure 4A), 45% decrease in aadA resistance gene expression (Figure 4B), and a corresponding 2-fold reduction in the spectinomycin MIC (Table 2). These results clearly demonstrate the strong positive regulatory role of pcnB in plasmid maintenance and resistance transmission. Thus, pcnB is essential for the high-level antibiotic resistance mediated by ColE1-type plasmids. This finding is highly consistent with recent studies in Escherichia coli, further supporting the conserved function of pcnB in plasmid biology among Enterobacteriaceae (Schubert et al., 2025; Wellner et al., 2025).
Particularly noteworthy is that the dual mechanisms of pcnB in regulating both plasmid copy number and mRNA stability explain its “contradictory” roles in gene expression regulation (Schubert et al., 2025). For specific chromosome-encoded genes, pcnB deletion increases their mRNA stability and expression levels by impairing polyadenylation-mediated RNA decay. Conversely, for plasmid-encoded genes, pcnB deletion decreases plasmid copy number by stabilizing antisense RNAs that inhibit replication, thereby indirectly reducing gene dosage. Although reduced plasmid copy number should theoretically decrease gene dosage, the concomitant loss of mRNA degradation function in ΔpcnB actually increases transcript stability for some plasmid genes (e.g., aadA), resulting in phenotypic changes smaller than copy number variations. This complex regulatory pattern suggests that pcnB occupies a central hub position in bacterial gene expression networks, fine-tuning gene expression levels by coordinating gene dosage and mRNA stability.
Furthermore, our study revealed that pcnB deletion significantly impaired biofilm formation capacity and overall bacterial viability (Figures 2A,B). As a critical virulence phenotype for bacterial colonization and environmental stress resistance, the weakened biofilm formation may be attributed to multiple factors (Guerra et al., 2022): first, the stability of quorum-sensing signaling molecules might decrease due to pcnB deletion (Sinha et al., 2018); second, bacteria may undergo metabolic energy redistribution; third, outer membrane integrity might be altered. Meanwhile, the reduced FDA hydrolysis activity further indicates widespread defects in enzymatic activity and metabolic states in ΔpcnB strains, which is highly consistent with their growth delay phenotype in minimal medium. These findings provide new experimental evidence for comprehensively understanding the regulatory role of pcnB in bacterial physiological metabolism. Although this study establishes the critical role of pcnB in virulence plasmid stability, we acknowledge a limitation: The exact mechanism involving the half-life of aadA mRNA remains to be elucidated in future work.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.
Author contributions
LZ: Visualization, Writing – original draft, Validation, Data curation, Investigation. SZ: Validation, Data curation, Writing – original draft, Investigation, Software. JW: Data curation, Writing – original draft. ZZ: Writing – original draft, Investigation. YX: Supervision, Methodology, Formal analysis, Conceptualization, Writing – review & editing. CZ: Funding acquisition, Conceptualization, Writing – review & editing, Resources, Project administration, Methodology.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This research was funded by a grant from the Natural Science Foundation for Universities of Anhui Province (KJ2020A0115).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2025.1709161/full#supplementary-material
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Keywords: Klebsiella pneumoniae , pcnB, siderophore, EPS, antibiotic resistance, biofilm
Citation: Zhang L, Zhang S, Wang J, Zhang Z, Xu Y and Zhang C (2025) The poly (A) polymerase pcnB modulates virulence and resistance in Klebsiella pneumoniae by differentially regulating chromosomal mRNA stability and plasmid copy number. Front. Microbiol. 16:1709161. doi: 10.3389/fmicb.2025.1709161
Edited by:
Muhammad Kamruzzaman, Westmead Institute for Medical Research, AustraliaReviewed by:
Kwok Jian Goh, Monash University, AustraliaAntônio Alexandre De Vasconcelos Júnior, Clementino Fraga laboratory
Copyright © 2025 Zhang, Zhang, Wang, Zhang, Xu and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yong Xu, eHV5b25nMTAxM0AxNjMuY29t; Chen Zhang, c3d6emMwMDFAMTYzLmNvbQ==