Inner membrane proteins play diverse important roles in Gram-negative bacteria, such as transportation of small molecules, electron transfer, sensing of extracellular signals and transduction, enzymatic reactions, formation of appendages, cytokinesis, and regulation. In particular, the regulatory mechanisms of the non-enzymatic inner membrane proteins are poorly understood.

LapB (formerly known as YciM), a lipopolysaccharide assembly protein B, is a non-enzymatic inner membrane protein whose N-terminal domain forms a transmembrane helix, and its C-terminal domain is unusually predicted to be located in the cytosol (Nicolaes et al., 2014). LapB regulates the protein level of LpxC, a soluble enzyme that catalyzes the second reaction of lipopolysaccharide (LPS) biosynthesis, through its role as an adaptor protein of the essential inner membrane protease FtsH which directly degrades LpxC (Klein et al., 2014; Mahalakshmi et al., 2014; Fivenson and Bernhardt, 2020; Guest et al., 2020). Recent studies have demonstrated that the LapB-mediated enhancement of LpxC proteolysis is inhibited by YejM, an essential inner membrane protein, through direct interaction with LapB (Cian et al., 2019; Biernacka et al., 2020; Fivenson and Bernhardt, 2020; Guest et al., 2020; Nguyen et al., 2020; Lee et al., 2021). In addition to the regulation of LpxC proteolysis, LapB appears to play other regulatory roles. The lapB mutant exhibited a strong sensitivity to cold stress, and this phenotype was not associated with LpxC (Lee et al., 2021).

YhcB is also a non-enzymatic inner membrane protein whose N-terminal domain forms a transmembrane helix, and its C-terminal domain is unusually predicted to be located in the cytosol (Sung et al., 2020; Mehla et al., 2021). Depletion of YhcB induces abnormal morphology and defects in cell division (Sung et al., 2020; Mehla et al., 2021), and YhcB interacts with various cell division-related proteins such as RodZ (Li et al., 2012; Mehla et al., 2021). A recent report using transposon mutagenesis and deep sequencing (Tn-seq) experiments showed that depletion of YhcB caused failure of coordinated peptidoglycan, LPS, and phospholipid biosynthesis, suggesting that YhcB may play a role in the junction of several envelope biosynthetic pathways (Goodall et al., 2021).

In this study, we identified several novel phenotypes of the lapB mutant and demonstrated that most of these phenotypes were suppressed by loss of penicillin-binding protein 1b (PBP1b) or YhcB, in an LpxC-independent manner. Both suppressor mutants retained their phenotypes, indicating that LapB acts upstream of PBP1b and YhcB. Therefore, these results suggest that LapB may function in the regulation of peptidoglycan biosynthesis as well as LpxC-mediated regulation of LPS biosynthesis.

Bacterial strains, plasmids, and culture conditions

All strains and plasmids used in this study are listed in Supplementary data Table S1 and all primers are presented in Supplementary data Table S2. Escherichia coli cells were cultured in lysogeny broth (LB) medium at 37°C, unless mentioned otherwise. Alkaline LB medium was prepared by adding 50 mM Tris (final concentration), followed by pH adjustment of the medium using a 10 N NaOH solution. Similarly, acidic LB medium was prepared by adding 50 mM sodium citrate (final concentration), followed by pH adjustment of the medium using a 10 N HCl solution. M9 minimal medium containing the indicated carbon sources was used as a defined medium. Alanine (final concentration of 10 mM), instead of ammonium salt, was used as a nitrogen source in the M9 minimal medium for nitrogen starvation. Antibiotics, including ampicillin (final concentration of 100 μg/ml), kanamycin (50 μg/ml), tetracycline (10 μg/ml), and chloramphenicol (5 μg/ml), were added to the culture medium when necessary.

Additional deletion of mrcA, mrcB, lpoA, lpoB, or yhcB gene in the lapB mutant was performed using λ red recombinase as previously described (Datsenko and Wanner, 2000; Kim et al., 2021). The kanamycin resistance gene of the pKD13 plasmid was amplified by polymerase chain reaction (PCR) using primers containing 50 nt flanking sequences homologous to the target gene. After PCR purification, the PCR product was transformed into lapB mutant cells harboring the pKD46 plasmid expressing λ red recombinase. The deletion mutant defective for the target gene was selected on LB plates containing kanamycin. The deletion of the target gene was confirmed using PCR. To remove the kanamycin resistance gene, the pCP20 plasmid expressing the FRT recombinase was transformed into a deletion mutant. The removal of the kanamycin resistance gene was confirmed by PCR, and the pCP20 plasmid was cured by incubation at 37°C.

The strain harboring the 3 × Flag gene in the chromosomal 3' region of mrcA, mrcB, or yhcB was also constructed using λ red recombinase, as previously described (Kim et al., 2021; Park et al., 2022). The 3 × Flag and kanamycin resistance genes of the plasmid pBAD-Flag-FRT-Kan were amplified by PCR using primers containing 50 nt flanking sequences homologous to the chromosomal 3' region of the target gene. The template plasmid was removed via overnight incubation with DpnI. After PCR purification, the PCR product was transformed into wild-type cells harboring the pKD46 plasmid. The mutant cells with the insertion of the 3 × Flag gene were selected on LB plates containing kanamycin. The insertion of the 3 × Flag gene into the chromosomal 3' region of the target gene was confirmed by PCR.

The pRE1-LapB and pRE1-LapB(ΔTM) plasmids expressing LapB and LapB without the transmembrane domain, respectively, were constructed previously (Lee et al., 2021). Since the lapB gene in the pRE1 plasmid is expressed under the control of the cI protein of λ phage, it is constitutively expressed in MG1655 cells lacking the cI protein (Reddy et al., 1989; Lee et al., 2015; Choi et al., 2016).

Determination of cell growth by spotting serial dilutions of the cells

To measure the growth of the bacterial cells under various stress conditions, cells grown at 37°C overnight were serially diluted from 10⁸ to 10⁴ cells/ml in LB or M9 minimal medium. A volume of 2 μl from the diluted samples was spotted onto the indicated plates. After incubation, until the wild-type cells from 10⁴ cells/ml samples formed colonies, pictures of the plates were taken using a digital camera EOS 100D (Canon Inc.).

Examination of cell shape using fluorescence microscopy

Cells grown in the LB medium at 37°C overnight were inoculated into the LB medium. When an OD_600nm was approximately 0.4 (approximately 4 × 10⁸ cells/ml), the cells were spotted on 1% agarose pads containing phosphate-buffered saline after staining with 5 μg/ml FM4-64 [N-(3-triethylammoniumpropyl) -4-(p-diethylaminophenylhexatrienyl)-pyridinium dibromide] at room temperature for 10 min. Cell morphology was observed using an Eclipse Ni microscope (Nikon).

Western blot analysis

Cells grown overnight at 37°C were inoculated into the LB medium. When an OD_600nm was approximately 0.4 (approximately 4 × 10⁸ cells/ml), the cells were harvested by centrifugation. Sodium dodecyl sulfate sample buffer was added to the cells and the mixtures were vigorously vortexed. After boiling for 5 min, 20 μl of the samples was loaded onto 4–20% Tris–glycine polyacrylamide gels (KOMA Biotech). After separation, total proteins in the polyacrylamide gel were transferred onto PVDF membranes. The protein levels of target proteins were determined using a monoclonal antibody against Flag-tag (Santa Cruz Biotechnology) and anti-LpxC antibody (Cusabio Biotech) according to the standard procedures, and anti-FtsZ (Agrisera) antibody was used to detect FtsZ as a loading control.

Determination of minimal inhibitory concentration (MIC)

The MIC of cefoxitin was measured according to the Clinical Laboratory Standards Institute guideline (CLSI, 2018). Cells grown overnight at 37°C were inoculated into Mueller-Hinton broth. The cells were cultured at 37°C to a McFarland turbidity standard of 0.5 (approximately 1.5 × 10⁸ cells/ml) and harvested by centrifugation. Harvested cells were diluted to a final concentration of 10⁷ cells/ml using Mueller-Hinton broth. A volume of 10 μl of diluted samples was spotted onto Mueller-Hinton plates containing cefoxitin at final concentrations of 512 μg/ml to 3.9 ng/ml in two-fold serial dilutions. After incubation at 37°C for 20 h, images of the plates were taken. The MIC value corresponds to the lowest concentration of cefoxitin at which the cells do not exhibit lawn growth.

Pleiotropic phenotypes of the lapB mutant

In a previous study (Lee et al., 2021), we showed that the lapB mutant is strongly sensitive to cold stress. Although LapB has been reported to be essential for growth under standard laboratory conditions (Mahalakshmi et al., 2014), in our experimental conditions, LapB was not essential and the identified phenotype of the lapB mutant was completely complemented by ectopic expression of LapB (Lee et al., 2021). To analyze the intracellular role of LapB, we searched other phenotypes of the lapB mutant, through measuring its cell growth under various stress conditions. As expected, the lapB mutant exhibited the strong growth defects under cold stress (Fig. 1A). Additionally, growths defect in the lapB mutant were observed under many other stress conditions. The lapB mutant barely grew when bile salt and ethanol were present in the LB medium or when it was exposed to alkaline growth conditions. This mutant was also slightly sensitive to salt, copper, and urea stresses. The lapB mutant showed a strong red color in the membrane permeability test using CPRG, a lactose derivative that can penetrate the envelope with increased permeability and is degraded to chlorophenyl red by intracellular β-galactosidase (Paradis-Bleau et al., 2014). This result indicates that the lapB mutant has increased membrane permeability.

Fig. 1. Pleiotropic phenotypes of the lapB mutant.
(A) Various phenotypes of the lapB mutant in complex media. The wild-type and lapB mutant cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto LB plates without or with indicated chemicals, or alkaline or acidic LB plates. Besides plates presenting incubation temperature, all plates were incubated at 37°C. (B) Various phenotypes of the lapB mutant in chemically defined media. The wild-type and lapB mutant cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto M9 minimal medium plates containing indicated carbon sources. M9(-NH₃) means M9 minimal medium without ammonium salt. All plates were incubated at 37°C.

We also examined the phenotypes of the lapB mutant on M9 minimal media. Although the lapB mutant showed growth comparable to that of the wild-type strain in M9 minimal media containing sugars as a carbon source, it showed severe growth inhibition in energy-poor carbon sources such as organic acid intermediates of the citric acid cycle (Fig. 1B). These phenotypes were also observed in liquid cultures. The lapB mutant did not grow in M9 minimal liquid medium containing α-ketoglutarate and fumarate as the carbon source (Fig. 2A). The growth retardations of the lapB mutant was complemented by ectopic expression of LapB (Fig. 2A). LapB has a transmembrane domain at its N-terminus and a previous report showed that the transmembrane domain of LapB is indispensable for its function (Lee et al., 2021). We examined whether the transmembrane domain of LapB is important for novel identified phenotypes. Expression of full-length LapB complemented all the tested phenotypes of the lapB mutant, whereas expression of truncated LapB without the transmembrane domain did not complement all the phenotypes tested (Fig. 2B). Therefore, these results demonstrate that deletion of the lapB gene causes pleiotropic phenotypes and that the transmembrane domain of LapB is necessary for its function.

Fig. 2. The transmembrane domain of LapB is indispensable for its function.
(A) Complementation of growth defects of the lapB mutant in M9 minimal media containing energy-poor carbon sources. The wild-type (closed circles and triangles), lapB mutant (open circles and triangles), and lapB mutant harboring pRE1-LapB (slashed circles and triangles) cells grown in LB medium overnight were inoculated in M9 minimal medium containing α-ketoglutarate (circles) or fumarate (triangles) as a carbon source. Cell growth was recorded by measuring the optical density at 600 nm. (B) Requirement of the transmembrane domain of LapB in complementation of the phenotypes of the lapB mutant. The indicated strain cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto LB plates under indicated stress conditions or M9 minimal medium plates containing 0.2% α-ketoglutarate and fumarate as a carbon source. Besides plates presenting incubation temperature, all plates were incubated at 37°C.

Depletion of PBP1b suppresses most phenotypes of the lapB mutant

Although the lapB mutant exhibited increased envelope permeability (Fig. 1A), this mutant strain was resistant to vancomycin, an antibiotic with a high molecular weight, to which cells with increased envelope permeability generally show increased susceptibility (Mitchell et al., 2018; Lee et al., 2021). Susceptibility to vancomycin can also be changed by alteration of peptidoglycan (Park et al., 2022). Therefore, we investigated whether LapB affects peptidoglycan synthesis. As alterations in peptidoglycan synthesis sometimes affect bacterial morphology (Mueller et al., 2019; Rohs and Bernhardt, 2021), we examined the morphology of the lapB mutant. In LB medium, some cells of the lapB mutant showed filamentous morphology (Supplementary data Fig. S1). Therefore, we tested the effect of PBPs on the phenotypes of the lapB mutant. Because PBP2 and PBP3 are essential (Rohs and Bernhardt, 2021), we observed the effect of the depletion of PBP1a (encoded by an mrcA gene) or PBP1b (encoded by an mrcB gene). Depletion of PBP1a did not affect all tested phenotypes of the lapB mutant, whereas depletion of PBP1b strongly suppressed most of the tested phenotypes, including sensitivity to bile salt and ethanol, growth defects under energy-poor carbon sources, and vancomycin resistance (Fig. 3A). Growth under alkaline stress was partially suppressed by the depletion of PBP1b. As increased protein level of LpxC in the lapB mutant was not changed by the deletion of the murB gene (Fig. 3B), these suppressions are not associated with the protein level of LpxC. Additionally, depletion of LpoB lipoprotein, an adaptor protein for the function of PBP1b, but not LpoA lipoprotein for the function of PBP1a, suppressed most phenotypes of the lapB mutant (Fig. 3C), confirming that PBP1b but not PBP1a is related to LapB. Surprisingly, the red color of the lapB mrcB double mutant was more intense than that of the lapB mutant in the CPRG experiment (Fig. 3A). Therefore, these results imply that many phenotypes of the lapB mutant may be associated with peptidoglycan synthesis but not with increased membrane permeability.

Fig. 3. Suppression of the phenotypes of the lapB mutant by depletion of PBP1b or LpoB.
(A) Suppression of the phenotypes of the lapB mutant by depletion of PBP1b. The indicated strain cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto LB plates under indicated stress conditions or M9 minimal medium plates containing indicated molecules as a carbon source. All plates were incubated at 37°C. (B) LpxC levels in the lapB mutant and lapB mrcB double mutant cells. The wild-type (1), lapB mutant (2), lapB mrcB double mutant (3), and MG1655/pBAD-LpxC (4) cells were grown in an LB medium containing 0.2% arabinose. Western blot analysis with anti-LpxC and anti-FtsZ antibodies was carried out using 2 × 10⁷ cells in MG1655/pBAD-LpxC and 2 × 10⁸ cells in other strains, harvested at the early exponential phase. The arrow indicates LpxC proteins. (C) Suppression of the phenotypes of the lapB mutant by depletion of LpoB. The indicated strain cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto LB plates without or with indicated chemicals, or an alkaline LB plate. Besides a plate presenting incubation temperature, all plates were incubated at 37°C.

Cefoxitin sensitivity of the mrcB mutant is conserved in the lapB mrcB double mutant

The mrcB mutant is strongly susceptible to several β-lactam antibiotics such as cefoxitin (Paradis-Bleau et al., 2010), but the exact mechanism is unknown. Because depletion of PBP1b suppressed most phenotypes of the lapB mutant, we wondered whether depletion of LapB affects the cefoxitin sensitivity of the mrcB mutant. The MIC of cefoxitin decreased 32-fold in the mrcB mutant compared to that in the wild-type cells (Fig. 4). The same MIC value was observed for the lapB mrcB double mutant. This result indicates that the depletion of LapB does not affect the phenotype of the mrcB mutant, which implies that LapB may be located functionally upstream of PBP1b.

Fig. 4. Cefoxitin sensitivity of the lapB mrcB double mutant.
The MICs of cefoxitin against the wild-type, mrcB mutant, lapB mrcB double mutant, and lapB mutant strains were measured in Mueller-Hinton broth plates containing cefoxitin of final concentrations of 512 μg/ml to 3.9 ng/ml in two-fold serial dilutions, according to the Clinical Laboratory Standards Institute guidelines. After incubation at 37°C for 20 h, pictures of the plates were taken.

Depletion of YhcB also suppresses most phenotypes of the lapB mutant

Our results imply that LapB may be associated with both LPS and peptidoglycan synthesis. A recent study showed that the depletion of the inner membrane protein YhcB induces dysregulation of peptidoglycan and LPS synthesis (Goodall et al., 2021). This report on YhcB prompted us to investigate whether YhcB affects LapB function. Notably, depletion of YhcB strongly suppressed all tested phenotypes of the lapB mutant (Fig. 5A). As increased protein level of LpxC in the lapB mutant was not changed by the deletion of the yhcB gene (Fig. 5B), these suppressions are also not associated with the protein level of LpxC, like PBP1b. It was noteworthy that depletion of YhcB significantly suppressed the alkaline sensitivity of the lapB mutant (Fig. 5A), unlike the slight suppression caused by depletion of PBP1b (Fig. 3A). Because the yhcB mutant exhibited a strong red color in an experiment using CPRG (Sung et al., 2020), we did not test the color of the lapB yhcB double mutant in the experiment using CPRG.

Fig. 5. Suppression of the phenotypes of the lapB mutant by depletion of YhcB.
(A) Suppression of most phenotypes of the lapB mutant by depletion of YhcB. The indicated strain cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto LB plates without or with indicated chemicals, an alkaline LB plate, or M9 minimal medium plates containing indicated molecules as a carbon source. Besides a plate presenting incubation temperature, all plates were incubated at 37°C. (B) LpxC levels in the lapB mutant and lapB yhcB double mutant cells. The wild-type (1), lapB mutant (2), lapB yhcB double mutant (3), and MG1655/pBAD-LpxC (4) cells were grown in an LB medium containing 0.2% arabinose. Western blot analysis with anti-LpxC and anti-FtsZ antibodies was carried out using 2 × 10⁷ cells in MG1655/pBAD-LpxC and 2 × 10⁸ cells in other strains, harvested at the early exponential phase. The arrow indicates LpxC proteins.

Most phenotypes of the yhcB mutant are conserved in the lapB yhcB double mutant

Similar to PBP1b, we wondered whether the depletion of LapB affects the phenotype of the yhcB mutant. The yhcB mutant was sensitive to vancomycin and SDS-EDTA stresses (Sung et al., 2020). In this study, we also found that the yhcB mutant was sensitive to bile salts (Fig. 6). Under all these stress conditions, the lapB yhcB double mutant phenocopied the yhcB mutant, but not the lapB mutant (Fig. 6). These results indicate that the depletion of LapB does not affect the phenotype of the yhcB mutant, which implies that LapB may be located functionally upstream of YhcB.

Fig. 6. Phenotypes of the lapB yhcB double mutant.
The indicated strain cells were serially diluted from 10⁸ cells/ml to 10⁴ cells/ml in 10-fold serial dilutions and spotted onto LB plates without or with indicated chemicals. Besides a plate presenting incubation temperature, all plates were incubated at 37°C.

LapB does not affect the protein levels of PBP1b and YhcB

LapB functions as an adaptor protein of the essential inner membrane protease FtsH and facilitates the FtsH-mediated proteolysis of LpxC, the cytoplasmic enzyme that catalyzes the first committed reaction of the LPS biosynthesis pathway (Klein et al., 2014; Mahalakshmi et al., 2014; Fivenson and Bernhardt, 2020; Guest et al., 2020). Our data revealed a functional relationship between LapB and PBP1b or YhcB. Therefore, we analyzed whether LapB regulates the protein levels of PBP1b and YhcB. To assess the protein levels of PBP1a, PBP1b, and YhcB, we constructed the strains with the insertion of a 3 × Flag gene into the chromosomal 3' region of mrcA, mrcB, and yhcB genes. The levels of the three proteins did not change in cells defective for LapB compared those in wild-type cells (Supplementary data Fig. S2), indicating that the protein levels of PBP1a, PBP1b, and YhcB are not regulated by LapB. Therefore, LapB seems to affect PBP1b and YhcB through other mechanisms, but not through the regulation of proteolysis.

The physiological role of LapB, an inner membrane protein involved in regulation, has not been definitively determined. Various studies have demonstrated that LapB positively regulates FtsH-mediated LpxC proteolysis by directly interacting with FtsH (Klein et al., 2014; Mahalakshmi et al., 2014; Cian et al., 2019; Biernacka et al., 2020; Fivenson and Bernhardt, 2020; Guest et al., 2020; Nguyen et al., 2020). However, the direct interaction between LapB and FtsH has been detected only in pull-down experiments (Klein et al., 2014), and has not been confirmed by other biochemical and genetic experiments. A recent report has shown that LapB may have an additional LpxC-independent role (Lee et al., 2021). In this study, we provided genetic evidence for the correlation between LapB and the regulation of peptidoglycan biosynthesis. We identified various novel phenotypes of the lapB mutant, most of which were suppressed by depletion of PBP1b and YhcB, inner membrane proteins involved in peptidoglycan biosynthesis, in an LpxC-independent manner. These results imply that many phenotypes of the lapB mutant may be caused by dysregulation of peptidoglycan biosynthesis.

Although PBP1a and PBP1b belong to class A PBP with both dd-transpeptidase and glycosyltransferase activities and contribute to peptidoglycan repair (Vigouroux et al., 2020), only depletion of PBP1b suppressed the phenotypes of the lapB mutant (Fig. 3). Various phenotypes, including β-lactam, acid, and salt sensitivities, have been observed in the mrcB mutant (Paradis-Bleau et al., 2010; Mueller et al., 2019; Park et al., 2020), whereas only the phenotype of the mrcA mutant is salt sensitivity (Park et al., 2020). To date, we do not know the exact reason why only the mrcB mutant suppresses the phenotype of the lapB mutant. However, these results suggest that LapB may be associated with the regulation of peptidoglycan synthesis.

Based on the phenotypes of the lapB mrcB and lapB yhcB double mutants, we suggest that LapB acts upstream of PBP1b and YhcB. Since the sole known molecular mechanism of LapB is the activation of LpxC proteolysis by FtsH, we examined the protein levels of PBP1b and YhcB in the lapB mutant. However, loss of LapB did not change the protein levels of PBP1b and YhcB (Supplementary data Fig. S2), indicating that LapB may affect these two proteins through other mechanisms, but not through FtsH-mediated proteolysis. A recent study showed that YhcB functions upstream of LapB and regulates LpxC proteolysis by directly interacting with LapB (Wieczorek et al., 2022). These results suggest that LapB and YhcB are affected bidirectionally. This should be more carefully investigated in future studies.

In this study, we demonstrated interesting novel phenotypes of the lapB mutant, including growth defects under energy-poor carbon sources and vancomycin resistance. Peptidoglycan biosynthesis requires the amino sugar, N-acetylglucosamine. Under energy-poor carbon sources, cells synthesize glucose through the gluconeogenesis pathway for peptidoglycan biosynthesis. Dysregulation of peptidoglycan biosynthesis by the loss of LapB could affect the bacterial growth under energy-poor carbon sources more strongly than under good carbon sources, such as glucose. An experiment using CPRG showed that the lapB mutant had increased membrane permeability (Fig. 1A). As vancomycin is an antibiotic with a high molecular weight, cells with increased membrane permeability are generally sensitive to vancomycin (O'Shea and Moser, 2008; Lee et al., 2017; Mitchell et al., 2018; Sung et al., 2020). However, the lapB mutant showed a strong resistance to vancomycin (Figs. 3 and 5). A recent report revealed that the amount of decoy D-alanine-D-alanine residues in peptidoglycan strongly affects vancomycin resistance (Park et al., 2022). Therefore, vancomycin resistance in the lapB mutant may be related to the amount of peptidoglycan biosynthesis but not membrane permeability.

내막단백질 LapB는 필수 막단백질인 FtsH에 의한 lipopolysaccharide (LPS) 합성 단백질 LpxC의 분해 조절에 참여한다. 그러나 lapB 결손 균주의 LpxC와 무관한 표현형의 존재는 LapB의 추가적인 생리적 기능이 존재함을 암시한다. 본 연구에서 우리는 대장균에서 LapB의 새로운 세포내 기능을 암시하는 유전적 증거를 제시한다. 우리는 저에너지 탄소원에서의 생장 저해 같은 lapB 결손 균주의 몇 가지 새로운 표현형을 찾았고, 이런 표현형이 펩티도글리칸 생합성에 관여하는 페니실린 결합 단백질 1b (PBP1b)의 결손에 의해 LpxC와 무관한 방식으로 회복되는 것을 발견하였다. 또한, lapB 결손 균주의 대부분의 표현형은 펩티도글리칸과 LPS의 조직적 생합성에 관여하는 내막 단백질인 YhcB의 결손으로도 역시 회복되었다. 종합적으로 본 실험 결과는 LapB가 LpxC에 의한 LPS 수준 조절에 관여할 뿐만 아니라 펩티도글리칸 생합성 조절에도 관여할 것임을 암시한다.

This work was supported by research grants from Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-RS-2023-00246684) and Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 322026-3).

Chang-Ro Lee is Editor of KJM. He was not involved in the review process of this article. Also, authors have no conflicts of interest to report.