Revisiting the Gram-Negative Lipoprotein Paradigm

Abstract

The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein N-acyltransferase. The mature lipoproteins are then sorted by the Lol system, with most Lpps inserted into the outer membrane (OM). We demonstrate here that the lnt gene is not essential to the Gram-negative pathogen Francisella tularensis subsp. tularensis strain Schu or to the live vaccine strain LVS. An LVS ⌬lnt mutant has a small-colony phenotype on sucrose medium and increased susceptibility to globomycin and rifampin. We provide data indicating that the OM lipoprotein Tul4A (LpnA) is diacylated but that it, and its paralog Tul4B (LpnB), still sort to the OM in the ⌬lnt mutant. We present a model in which the Lol sorting pathway of Francisella has a modified ABC transporter system that is capable of recognizing and sorting both triacylated and diacylated lipoproteins, and we show that this modified system is present in many other Gram-negative bacteria. We examined this model using Neisseria gonorrhoeae, which has the same Lol architecture as that of Francisella, and found that the lnt gene is not essential in this organism. This work suggests that Gram-negative bacteria fall into two groups, one in which full lipoprotein processing is essential and one in which the final acylation step is not essential, potentially due to the ability of the Lol sorting pathway in these bacteria to sort immature apolipoproteins to the OM. IMPORTANCEThis paper describes the novel finding that the final stage in lipoprotein processing (normally considered an essential process) is not required by Francisella tularensis or Neisseria gonorrhoeae. The paper provides a potential reason for this and shows that it may be widespread in other Gram-negative bacteria.

A More Flexible Lipoprotein Sorting Pathway

“…LoVullo and colleagues confirmed this by constructing ⌬lnt deletion mutations in two different F. tularensis strains, including a human-pathogenic F. tularensis subsp. tular-ensis strain (5). The study further demonstrated that the known F. tularensis lipoprotein Tul4 (LpnA) shifts from a triacylated form in the wild-type strain to a diacylated form in the ⌬lnt background and that Tul4 and additional lipoproteins are still properly sorted to the OM in the ⌬lnt mutant.…”

“…Interestingly, the Lol system of F. tularensis lacks a gene for LolE, which in E. coli heterodimerizes with LolC to form the membrane component of the ABC transporter complex. Comparison of the lol genes present in various Gram-negative bacteria revealed that the absence of lolE is not unique to Francisella, but instead was found in more than half of the bacterial genomes they analyzed (5). Based on protein sequence analysis, LoVullo et al concluded that the single LolC present in bacteria such as Francisella spp.…”

“…Due to the high cost associated with lipoprotein mislocalization, Gram-negative bacteria have evolved a conserved mechanism for the processing and sorting of these molecules, to ensure they correctly reach their final destination. In this issue of the Journal of Bacteriology, LoVullo et al (5) challenge the current paradigm for lipoprotein processing and sorting in Gram-negative bacteria.…”

“…LoVullo et al (5) hypothesized that the Lol pathway of Francisella must contain some alternative functionality that allows it to recognize diacylated lipoproteins. Interestingly, the Lol system of F. tularensis lacks a gene for LolE, which in E. coli heterodimerizes with LolC to form the membrane component of the ABC transporter complex.…”

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

“…42 It is also possible that the presence of LolE permease, part of ABC transporter LolCDE, on E. coli contributed to resistance to this set of compounds, while the other Gram-negative F. tularensis was greatly inhibited. 43 In support of this, one group examining SMIs opted to use a strain of E. coli that has a shortened LPS, which renders it more permeable to SMIs. 44 This technique will be worth considering in future studies.…”