Elucidating the Functions of fabF1 and fabF2 in Pseudomonas aeruginosa: Implications for Fatty Acid Metabolism and Pathogenicity

doi:10.21203/rs.3.rs-5286450/v1

Background

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe infections in immunocompromised individuals, including burn patients and those with cystic fibrosis. β-ketoacyl-ACP synthases are a class of key enzymes in bacterial fatty acid metabolism, with functions that directly impact basic cellular metabolism and pathogenicity. Two types of long-chain β-ketoacyl-ACP synthases have been identified: FabB and FabF. This study investigates the roles of fabF1 and fabF2 genes in the fatty acid biosynthesis and virulence of P. aeruginosa PAO1.

Results

Complementation assays in Escherichia coli demonstrated that fabF2can substitute for the E. coli FabB enzyme, while FabF1 exhibits FabF-like activity. In P. aeruginosa PAO1, deletion of fabF1significantly decreased cis-vaccenic acid levels and increased palmitoleic acid, whereas deletion of fabF2 had no effect. The double mutant showed a marked reduction in cis-vaccenic acid. Virulence assays revealed that the ΔfabF1strain exhibited a 63% reduction in rhamnolipid production, while the ΔfabF2strain showed a 45% reduction. The double mutant retained only 28% of wild-type rhamnolipid levels. Additionally, pyoverdine secretion was substantially reduced in the double mutant, and both LasA protease activity and pyocyanin production were compromised. Motility assays indicated reduced swimming, twitching, and swarming abilities in the mutants.

Conclusions

These findings underscore the crucial roles of fabF1 and fabF2 in the fatty acid biosynthesis, virulence factor production, and motility of P. aeruginosa, providing insights into potential targets for antimicrobial development.

P. aeruginosa

fabF

ketoacyl-ACP synthase

fatty acid metabolism

motility

Pseudomonas aeruginosa is an opportunistic pathogen in humans, frequently associated with infections in burn patients and the respiratory tracts of individuals with cystic fibrosis. It has emerged as a significant nosocomial pathogen globally and as an invader in individuals with compromised immunity, such as malnourished children and people living with HIV [1, 2]. The infection of P. aeruginosa is facilitated by Type III secretion systems, biofilm formation, and various virulence factors, allowing it to effectively colonize and damage host tissues[3]. Fatty acids play a crucial role in both maintaining bacterial viability and enhancing virulence. Most membrane phospholipids are glycolipids containing two fatty acid chains, making fatty acids major components of cell membranes[4, 5, 6]. The long-chain ketoacyl-ACP synthase FabB and FabF in bacterial fatty acid synthesis system (FAS II) are considered to be important target sites for the development of novel antimicrobial drugs[5, 7, 8, 9] .

In Escherichia coli, 3-ketoacyl-ACP synthase I (FabB) catalyzes the elongation of cis-3-decenoyl-ACP to long-chain unsaturated acyl-ACPs, making it a key enzyme in unsaturated fatty acid (UFA) synthesis. Additionally, 3-ketoacyl-ACP synthase II (FabF) exhibits high catalytic activity in the conversion of cis-9-hexadecenoyl-ACP (palmitoleoyl-ACP) to cis-11-octadecenoyl-ACP (cis-vaccenoyl-ACP) [10, 11, 12]. Inactivation of FabF results in a significant decrease in the elongation of shorter unsaturated fatty acids in the phospholipids of E. coli as well as Photobacterium profundum and Pseudomonas putida[13, 14]. However, cis-vaccenoyl-ACP remains in the PAO1 FabF strain, suggesting the presence of a second 3-ketoacyl-ACP synthase in P. aeruginosa [15, 16].

Three genes were annotated as fabF in the P. aeruginosa PAO1 genome. The fabF1 gene is located in the fatty acid synthesis gene cluster. fabF3 (PA5174), which can utilize short-chain acetyl-CoA in the initial steps of de novo fatty acid biosynthesis, is believed to be the true 3-ketoacyl-ACP synthase III (FabH) in P. aeruginosa[17, 18]. FabF2, encoded by PA1373 and located in a separate operon, shares 46.2% identity with E. coli FabF. In this work, we explored the function of the P. aeruginosa fabF2 gene and its product.

Bacterial Strains, Plasmids, and Growth Conditions

The bacterial strains and plasmids employed in this study are detailed in Table 1. E. coli strains were cultured at 30°C, 37°C, or 42°C, depending on experimental requirements. For routine culturing of both E. coli and P. aeruginosa, Luria-Bertani (LB) medium, consisting of 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl (pH 7.0), was used. To assess the growth of temperature-sensitive (Ts) strains, RB medium, comprising 10 g/L tryptone, 1 g/L yeast extract, and 10 g/L NaCl (pH 7.0), was utilized. The following antibiotics were added at specified concentrations (µg/mL) where required: ampicillin (100 for E. coli), kanamycin sulfate (30), chloramphenicol (30), and gentamicin (10 for E. coli or 100 for P. aeruginosa). L-arabinose was used for induction at a final concentration of 0.01%, and IPTG was employed at a final concentration of 1 mM.

Table 1

Strains and plasmids in this work
Strains	Genotype		Sources
E. coli CY244	fabB, fabF(Ts)		Lab stock
E. coli CL28	∆fabF		Lab stock
E. coli K1060	∆fabB		Lab stock
BL21(DE3)	λ(DE3[lacI lacUV5-T7 gene1 ind1 sam7 nin5])		Lab stock
P. aeruginosa PAO1	Wild type		Lab stock
P. aeruginosa Zl206	∆fabF1		This work
P. aeruginosa Zl207	∆fabF2		This work
P. aeruginosa Zl208	∆fabF1, ∆fabF2		This work
P. aeruginosa Zl209	pafabF2-pSRK/Zl207		This work
Plasmids	Back bone	Gene	Sources
pBAD24M	Vector		Lab stock
pET-28b	Vector		Lab stock
pK18mobSacB	Vector		Lab stock
pSRK(Gm)	Vector		Lab stock
pZH31	pET-28b	E.coli fabA	Lab stock
pZH32	pET-28b	E.coli fabB	Lab stock
pZH33	pET-28b	E.coli fabD	Lab stock
pZH36	pET-28b	E.coli fabG	Lab stock
pYFJ84	pET-16a	V. harveyi AasS	Lab stock
pZL201	pBAD24M	P. aeruginosa fabF1	This work
pZL202	pBAD24M	P. aeruginosa fabF2	This work
pZL203	pBAD24M	P. aeruginosa fabB	This work
pZL204	pET-28b	P. aeruginosa fabF1	This work
pZL205	pET-28b	P. aeruginosa fabF2	This work
pZL206	pSRK(Gm)	P. aeruginosa fabF2	This work
pZL207	pK18mobSacB	P. aeruginosa fabF1UD-Gm	This work
pZL208	pK18mobSacB	P. aeruginosa fabF2 UD-Gm	This work

Recombinant DNA Techniques and Plasmid Construction

To clone the fabF1 and fabF2 genes from P. aeruginosa, genomic DNA was isolated from the PAO1 strain using the Takara DNA extraction kit. The target genes were amplified via PCR using Pfu DNA polymerase, and the resulting PCR products were digested with NdeI and HindIII. These fragments were then ligated into the pBAD24M vector at the corresponding restriction sites, resulting in the construction of plasmids pBAD24M-fabF1 (pZL201), pBAD24M-fabF2 (pZL202), and pBAD24M-fabB (pZL203). Similarly, the plasmids pSRK-fabF1 (pZL209), pSRK-fabF2 (pZL206), pET-28b-fabF1 (pZL204), and pET-28b-fabF2 (pZL205) were constructed using the same procedure. All constructs were verified by DNA sequencing, performed by Shanghai Sangon, Inc. (Shanghai, China).

Mutant strains of P. aeruginosa lacking fabF1 and fabF2 were generated through DNA double-crossover recombination, following the method described by Zhu [2]. Genetically complemented strains were created by transforming the mutant strains with the fabF genes carried on the pSRK vector. However, transformation of pSRK-fabF1 (pZL209) into the fabF1 mutant strain was unsuccessful.

Expression and Purification of Plasmid-Encoded Proteins

The plasmids pET-28b-fabF1 and pET-28b-fabF2 were introduced into E. coli strain BL21(DE3) for protein expression. The corresponding proteins, PaFabF1 and PaFabF2, were successfully expressed and subsequently purified. In parallel, the plasmid pYFJ84 was transformed into BL21(DE3) to express Vibrio harveyi AasS protein. AasS purification followed the protocol outlined by Jiang [19].

Motility Assay

The motility assays were conducted following the procedure described by Huang[20]. To assess swarming motility in P. aeruginosa, the following medium was prepared: 0.45% tryptone, 0.13% yeast extract, 0.5% agarose, 0.22% NaCl, and 0.5% agar. Before use, plates were allowed to air-dry for 5–10 minutes. Bacterial cells were grown to an OD₆₀₀ of 0.8 and inoculated gently at the center of the plate using a toothpick. The plates were incubated at 30°C for 24 hours.

Twitching motility was examined using a medium consisting of 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 1% agarose. The bacterial cells were stabbed to the bottom of the agar plate using a toothpick, and the plates were incubated at 37°C for 20 hours. Colony movement at the interface between the agar and the petri dish was observed to assess twitching motility.

Siderophore Secretion Assay

Chromazurol S (CAS)-LB plates were prepared using a modified version of a previously established method. A 10× blue dye solution, composed of 1 mM chromeazurol S (CAS) (Acros), 2 mM cetyltrimethylammonium bromide, and 500 µM FeCl₃, was sterilized by autoclaving. After sterilization, 10 mL of this blue dye solution was mixed with 100 mL of molten LB Miller agar (1.5%). Once the plates had solidified, they were air-dried at room temperature for 1 hour before streaking bacterial cultures to test siderophore secretion by each strain.

LasA Activity Assay

P. aeruginosa cells were cultured in LB medium at 37°C for 12 hours. Following incubation, 10 mL of the culture was centrifuged at 5,000 ×g for 10 minutes at 4°C to remove the cells. The LasA protease activity was assessed by measuring the ability of the culture supernatant to lyse boiled Staphylococcus aureus RN4200 cells. An overnight culture of S. aureus grown in tryptic soy broth (30 mL) was boiled for 10 minutes, then centrifuged at 10,000 ×g for 10 minutes. The pellet was resuspended in 10 mM Na₂HPO₄ (pH 7.5) to an OD₆₀₀ of 0.9. To measure LasA activity, 100 µL of P. aeruginosa supernatant was added to 900 µL of S. aureus suspension, and the OD₆₀₀ was recorded at intervals of 5, 10, 15, 20, 25, 30, 35, 40, 45, 60, 75, 90, and 105 minutes.

Pyocyanin Quantitation Assay

The quantification of pyocyanin was performed by measuring its absorbance at 520 nm in an acidic environment. A 5-mL culture sample grown in LB medium was subjected to extraction with 3 mL of chloroform. The pyocyanin-containing chloroform phase was then re-extracted with 1 mL of 0.2 N HCl, resulting in a pink to deep red aqueous solution. The absorbance of this solution was recorded at 520 nm. Pyocyanin concentration in the culture supernatant was calculated by multiplying the absorbance at 520 nm (OD520) by the conversion factor 17.072, yielding values expressed as micrograms (µg) of pyocyanin per milliliter of supernatant.

Rhamnolipid Identification

Rhamnolipids were extracted from P. aeruginosa cultures grown for 24 hours at 30°C in PPGAS medium (containing 20 mM NH4Cl, 20 mM KCl, 120 mM Tris-HCl at pH 7.2, 1.6 mM MgSO4, 0.5% glucose, and 1.0% peptone). Cells were pelleted by centrifugation (10 minutes at 6,000 × g), and the supernatant was acidified to pH 2 with concentrated HCl. Equal volumes (1 mL) of the acidified supernatant and chloroform(2:1) were vortexed for 1 minute, followed by centrifugation at 10,000 × g for 10 minutes. The lower organic phase was collected, and the extraction process was repeated. The pooled organic layers were dried under vacuum, re-suspended in 1 mL methanol, filtered through a 0.45-µm membrane, evaporated to dryness, and finally re-dissolved in 20 µL methanol.

Thin-layer chromatography (TLC) was performed using silica 60 F254 plates (Merck, Darmstadt, Germany) with chloroform:methanol acid (65:15:2, v/v/v) as the mobile phase. Rhamnolipid spots were visualized by staining with orcinol reagent dissolved in 15% H2SO4 at a final concentration of 2%, followed by heating at 100°C for 2–5 minutes. Rhamnolipid concentration was determined using the sulfuric acid-anthrone method (0.2% anthrone, 85% sulfuric acid), with rhamnose as a standard, measuring absorbance at 620 nm.

To identify the fatty acid moieties of the rhamnolipids, 5 mL of supernatant was extracted as described above, and fatty acid methyl esters were synthesized. Briefly, rhamnolipids were dissolved in 1.2 mL of dry methanol, and 0.2 mL of 25% sodium methoxide (vol/vol) was added. After 15 minutes at room temperature, 1.2 mL of 2 M HCl was added, and fatty acid methyl esters were extracted by three successive extractions with 1.2 mL of petroleum ether. The solvent was evaporated under nitrogen, and the residues were analyzed for hydroxylacyl-ACP formation. The reaction mixture contained 0.1 M sodium phosphate (pH 7.0), 10 mM ATP, 20 mM MgSO4, 1 mM DTT, 100 µM E. coli ACP, 1 µg/µL of His-tagged V. harveyi AasS, and fatty acid methyl esters derived from rhamnolipid. The mixture was incubated at 37°C for 1 hour and resolved using conformationally sensitive gel electrophoresis on 17.5% polyacrylamide gels with 2.5 M urea for optimal separation.

Complementation of E. coli fabB and fabF Mutants by Genes Encoding P. aeruginosa FabF2 Proteins

To test the activities of P. aeruginosa fabF1 and fabF2 genes, we used the E. coli strain CY244 (ΔfabB, fabF(Ts)), which lacks 3-ketoacyl-ACP synthase II and 3-ketoacyl-ACP synthase I activity at the nonpermissive temperature[21]. Due to this deficiency, strain CY244 was unable to synthesize saturated fatty acids and cannot grow at 42°C, even when supplemented with oleic acid. We inserted the P. aeruginosa fabF1, fabF2, and fabB genes into the vector pBAD24M and transformed the resulting plasmids into CY244. Growth of the transformants was tested on RB medium plates with or without oleic acid. Expression of fabF2 and fabB supported robust growth of the E. coli strain CY244 in the absence and presence of oleic acid at 42°C. In contrast, fabF1 expression supported growth only with the addition of oleic acid at the nonpermissive temperature (Fig. 1A). These data indicate that P. aeruginosa FabB and FabF2 possess the activity of E. coli FabB, whereas FabF1 exhibits the activity of E. coli FabF.

To confirm these results, we used the E. coli fabB mutant strain K1060, which is auxotrophic for unsaturated fatty acids (UFAs) at all growth temperatures due to a site mutation in fabB. Plasmids expressing P. aeruginosa fabB or fabF2 restored the growth of strain K1060 in the absence of oleic acid, further indicating that P. aeruginosa FabB and FabF2 can functionally replace E. coli FabB (Fig. 1B).

We also tested the E. coli strain CL28, a ∆fabF strain defective in the synthesis of cis-11-octadecenoyl-ACP, to determine whether P. aeruginosa FabF1, FabF2, and FabB have E. coli FabF activity[22]. The plasmids carrying P. aeruginosa fabF1, fabF2 or fabB were transformed into strain CL28, and the fatty acid compositions of the resulting strains were analyzed by GC-MS. High levels of expression of either P. aeruginosa fabF1 or fabF2 increased the cis-11-octadecenoate (cis-vaccenate) content of strain CL28, indicating that fabF1 and fabF2 both possess E. coli FabF function and can convert cis-9-hexadecenoyl-ACP to cis-11-octadecenoyl-ACP (Table 2).

Table 2

Analysis of fatty acid composition in CL28 strains
	pBAD24M	pZL203	pZL201	pZL202
n-C_14:0-3-OH	15.17 ± 2.29	10.56 ± 0.73	14.18 ± 0.89	11.85 ± 0.52
n-C_14:0	7.14 ± 0.37	11.35 ± 0.42	6.54 ± 0.82	12.06 ± 0.73
n-C_16:0	0.96 ± 0.08	0.27 ± 0.01	0.93 ± 0.01	0.29 ± 0.02
n-C_16:1	0.70 ± 0.11	0.34 ± 0.03	0.71 ± 0.09	0.68 ± 0.18
n-C_17:0	2.19 ± 0.56	15.73 ± 1.09	2.65 ± 0.32	10.25 ± 1.59
n-C_18:1	61.51 ± 1.48	41.96 ± 0.40	63.45 ± 0.82	46.89 ± 1.17
n-C_18:0	2.83 ± 0.17	2.77 ± 0.42	3.05 ± 0.07	2.85 ± 0.26

Analysis of P. aeruginosa FabF1, FabB and FabF2 Activities in vitro

P. aeruginosa fabF1 and fabF2 genes were expressed in E. coli strain BL21 (DE3) as described in the Materials and Methods section. The histidine-tagged fusion proteins were purified using nickel chelate chromatography. Denaturing gel electrophoresis confirmed that the purified FabF1 and FabF2 proteins had monomeric molecular masses of 44 kDa, consistent with the values calculated from the sequences of the tagged proteins (Fig. 2A). Tryptic peptide mass spectral analyses further confirmed the identities of the purified proteins.

To assay the activities of FabF1 and FabF2, we used similar methods to purify the E. coli fatty acid biosynthetic proteins FabB, FabD, FabA, and FabG, along with V. harveyi AasS (acyl-ACP synthetase). E. coli holo-ACP was also purified. The elongation steps of the fatty acid synthesis reaction were reconstituted in vitro, followed by analysis using conformationally sensitive gel electrophoresis. FabF1 and FabF2 were able to elongate octanoyl-ACP using malonyl-ACP to generate low levels of long-chain acyl-ACP species, similar to EcFabB (E. coli FabB). In the presence of 3-ketoacyl-ACP synthase I (EcFabB) or II (FabF1 and FabF2), octanoyl-ACP was elongated with malonyl-ACP to form 3-keto-decenoyl-ACP, which was then reduced by EcFabG to 3-hydroxydecenoyl-ACP. EcFabA subsequently converted 3-hydroxydecenoyl-ACP to trans-2-decenoyl-ACP. Since no enoyl-ACP reductase (such as EcFabI) was added, the fatty acid chain could not be further elongated ( Fig. 2B).

Construction of P. aeruginosa PAO1 fabF Deletions and Analysis of Their Phenotypes

To determine the physiological functions of the P. aeruginosa PAO1 fabF proteins in fatty acid biosynthesis, strains with deletions in each fabF gene were constructed via allelic replacement. The pK18Gm plasmid containing fabF1 or fabF2 knockout cassette (pZL207 and pZL208) was constructed and introduced into PAO1 by conjugation, as described in the “Experimental Procedures”. The ΔfabF1 ΔfabF2 double mutant was constructed using the same method. Genetically complementary strains for ΔfabF2 was constructed, but pSRK-fabF1(pZL209) was failed to transform into fabF1 mutant strains. All strains grew well in LB medium or M9 medium, indicating that the absence of any fabF gene did not affect overall growth (data not shown).

The fatty acid compositions of the fabF mutant strains were determined by GC-MS (Table 3). Compared to the wild-type strain, the ΔfabF1 strain showed a significant decrease in cis-vaccenic acid (C18:1) with a concomitant increase in palmitoleic acid (C16:1). Cis-vaccenic acid decreased from 47.78–23.82%, while palmitoleic acid increased from 18.81–35.13%. The fatty acid composition of the ΔfabF2 strain was indistinguishable from that of the wild-type strain. The ΔfabF1 ΔfabF2 double mutant maintained only 11.1% of cis-vaccenic acid.

Table 3

**Analysis of fatty acid composition in** **P. aeruginosa fabFs** **mutant strains**
	WT	Zl206	Zl207	Zl208
n-C_14:0	0.66 ± 0.37	0.91 ± 0.22	0.67 ± 0.22	NR
n-C_16:0	28.40 ± 508	33.33 ± 4.62	31.15 ± 6.51	43.7 ± 1.02
n-C_16:1	18.81 ± 2.11	35.13 ± 3.22	17.67 ± 2.44	39.94 ± 10.28
n-C_17:0	2.61 ± 0.44	4.71 ± 1.19	3.57 ± 1.11	2.93 ± 2.02
n-C_18:1	47.78 ± 3.32	23.82 ± 6.40	45.30 ± 5.02	11.17 ± 3.17
n-C_18:0	1.74 ± 0.22	2.40 ± 0.38	1.64 ± 1.02	2.38 ± 0.26

fabF 1 and fabF2 Genes are Involved in the Virulence of P. aeruginosa

In P. aeruginosa, fatty acids are utilized not only as essential components of membrane phospholipids but also for the production of rhamnolipids and the siderophore pyoverdine. All strains were tested for the production of rhamnolipids and pyoverdine as described in the Methods section. Rhamnolipids are formed by acylation of L-rhamnose with 3-hydroxydecanoyl-ACP, an intermediate of the fatty acid biosynthesis system. The levels of rhamnolipids produced by the P. aeruginosa fabF mutant strains were examined. The amount of rhamnolipids in the ΔfabF1 strain decreased by 63% compared to the wild-type PAO1. The mutation of fabF2 resulted in a decrease of 45%. Complementation with plasmid pZL206 did not restore the ability to produce rhamnolipids. Instead, the production in strain ZL208 was only 38% of the PAO1 level. The ΔfabF1 ΔfabF2 double mutant maintained only 28% of the rhamnolipid production of PAO1 (Fig. 3A).

Pyoverdine is the dominant siderophore of P. aeruginosa and is assembled from tetradecanoyl-ACP. Secretion of pyoverdine by the P. aeruginosa fabF mutant strains was examined by culturing on LB–chromeazurol S (CAS) indicator plates. Zones around the cultures that change from blue to yellow-orange indicate where the siderophores have sequestered Fe³⁺ away from the blue CAS-Fe³⁺ complex. Both the wild-type strain PAO1 and the ΔfabF2 strain generated similar prominent clear zones, suggesting that the mutation of fabF2 did not affect siderophore secretion. Interestingly, complementation of fabF2 with plasmid pZL206 in the ΔfabF2 strain (strain ZL208) decreased the diameter of the yellow-orange ring by 46% compared to the wild-type PAO1. The ΔfabF1 strain produced a yellow-orange ring as large as that of strain ZL208. Moreover, the ΔfabF1 ΔfabF2 double mutant maintained only 34% of the diameter of the wild-type PAO1 (Fig. 3D).

LasA protease activity was determined by measuring the ability of P. aeruginosa culture supernatants to lyse boiled S. aureus cells. The data showed that the activity of LasA in the mutant strains ΔfabF1, ΔfabF2, and ZL208 was almost the same as in the wild-type PAO1. More than 80% of the boiled S. aureus cells were lysed in 30 minutes, except in the double mutant strain. However, the supernatants of the ΔfabF1 ΔfabF2 double mutant strain could only lyse 50.7% of the boiled S. aureus cells after 105 minutes of treatment (Fig. 3C).

Pyocyanin is a phenazine secondary metabolite secreted during infection of the host. Pyocyanin imparts a green color to P. aeruginosa cultures; thus, all mutants incubated in liquid LB medium showed a reduction in green color compared to the wild-type, except the complementation strain ZL208 (data not shown). The production of pyocyanin in all mutant strains was then quantified. The level of pyocyanin produced by the ΔfabF1 strain was reduced by 20.9% compared to the wild-type PAO1. The ΔfabF1 ΔfabF2 double mutant strain showed a 39.3% reduction in pyocyanin production compared to the wild-type. The lack of PafabF2 led to a 7.47% decrease in pyocyanin production, but complementation of PafabF2 restored both the visual and quantitative levels of pyocyanin to wild-type levels (Fig. 3B).

Three types of motility—swimming, twitching, and swarming—were tested in all strains. The twitching pattern of the ΔfabF2 strain was almost the same as that of the wild-type PAO1. Surprisingly, the complementation strain ZL208 showed reduced twitching ability. The mutation of fabF1 decreased the twitching ability of P. aeruginosa, and the double mutant strain exhibited an even smaller twitching pattern than all other strains. Generally, the swimming pattern mirrored the twitching pattern. The ΔfabF1 and ΔfabF2 strains had smaller swimming zones than the PAO1 strain. Instead of restoring the swimming ability of the ΔfabF2 strain, the complementation strain ZL208 exhibited an even lower swimming ability, while the double mutant completely lost swimming ability. Next, swarming motility was tested on semisolid plates (0.5% agar). Wild-type PAO1 formed a typical swarming pattern. However, deletion of fabF1 caused P. aeruginosa to form a small circular lawn. The circular lawn of the ΔfabF2 strain was also smaller than that of PAO1 but larger than that of ΔfabF1. Moreover, the complementation strain ZL208 could restore swarming ability. The swarming motility of the double mutant was reduced to the same level of ΔfabF1 (Fig. 4).

Our study provides comprehensive insights into the roles of fabF1 and fabF2 genes in the fatty acid biosynthesis and virulence of P. aeruginosa PAO1. The complementation assays in E. coli revealed that fabF2 can effectively substitute for E. coli FabB, while FabF1 shows FabF-like activity. This suggests that fabF2 plays a significant role in the early steps of fatty acid elongation, similar to FabB in E. coli. The ability of fabF1 to exhibit FabF-like activity indicates its involvement in the elongation cycle, contributing to the diversity of fatty acids produced.

The fatty acid composition analysis of P. aeruginosa PAO1 mutants underscores the importance of fabF1 in maintaining cis-vaccenic acid levels. The significant decrease in cis-vaccenic acid and the concomitant increase in palmitoleic acid in the ΔfabF1 strain suggest that fabF1 is pivotal for the synthesis of longer-chain unsaturated fatty acids. In contrast, the ΔfabF2 strain exhibited no significant changes in fatty acid composition, indicating a more specialized or redundant role for fabF2 under the conditions tested. The double mutant's drastic reduction in cis-vaccenic acid further highlights the non-redundant functions of fabF1 and fabF2 in fatty acid biosynthesis[23, 24].

Virulence factor production assays demonstrated the impact of fabF deletions on the pathogenicity of P. aeruginosa. The marked reduction in rhamnolipid and pyoverdine production in the ΔfabF1 and double mutants underscores the role of fabF1 in virulence [25]. The reduction in rhamnolipid production affects biofilm formation and surface motility, crucial for colonization and infection. Pyoverdine, a key siderophore, is essential for iron acquisition in iron-limited environments, and its decreased production in the double mutant suggests impaired virulence[26, 27, 28].

LasA protease activity assays showed that the double mutant had significantly reduced proteolytic activity, which could compromise the bacterium's ability to degrade host tissues and evade immune responses. Similarly, the reduction in pyocyanin production, a key virulence factor and signaling molecule, in the ΔfabF1 and double mutants, indicates the broader impact of fabF genes on the regulatory networks governing virulence[29, 30].

Motility assays revealed that deletions in fabF1 and fabF2 impaired swimming, twitching, and swarming motilities, with the double mutant showing the most pronounced defects. These motility traits are critical for surface colonization, biofilm formation, and dissemination within the host. The impaired motility in the mutants suggests that fabF genes are integral to the coordination of cellular processes required for effective movement and colonization [31, 32].

In conclusion, our study elucidates the essential roles of fabF1 and fabF2 in the fatty acid biosynthesis and virulence of P. aeruginosa. The differential roles of these genes in lipid composition and pathogenicity highlight their potential as targets for novel antimicrobial therapies. Further research into the regulatory mechanisms governing fabF expression and their interaction with other metabolic and virulence pathways will provide deeper insights into the multifaceted role of fatty acid biosynthesis in bacterial pathogenicity.

This study reports the functions of the P. aeruginosa genes fabF1 and fabF2 in fatty acid synthesis metabolism and elucidates their impact on the pathogenicity, providing insights into potential targets for antimicrobial development.

Clinical trial number: not applicable.

Ethics approval and consent to participate: not applicable.

Consent for publication：All authors agree to publication

Availability of data and material： All data and materials in this paper are availability

Funding: Youth Program of Natural Science Foundation of Shandong province of China (ZR2020QC004), and grant from National Natural Science Foundation of China (32170052).

Competing interests: not applicable.

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No competing interests reported.

Elucidating the Functions of fabF1 and fabF2 in Pseudomonas aeruginosa: Implications for Fatty Acid Metabolism and Pathogenicity

Status:

Version 1

Abstract

Figures

Background

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions

Recombinant DNA Techniques and Plasmid Construction

Expression and Purification of Plasmid-Encoded Proteins

Motility Assay

Siderophore Secretion Assay

LasA Activity Assay

Pyocyanin Quantitation Assay

Rhamnolipid Identification

Result

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1