Localization of the Outer Membrane Protein OmpA2 in Caulobacter crescentus Depends on the Position of the Gene in the Chromosome

Abstract

The outer membrane of Gram-negative bacteria is an essential structure involved in nutrient uptake, protection against harmful substances, and cell growth. Different proteins keep the outer membrane from blebbing out by simultaneously interacting with it and with the cell wall. These proteins have been mainly studied in enterobacteria, where OmpA and the Braun and Pal lipoproteins stabilize the outer membrane. Some degree of functional redundancy exists between these proteins, since none of them is essential but the absence of two of them results in a severe phenotype. Caulobacter crescentus has a different strategy to maintain its outer membrane, since it lacks the Braun lipoprotein and Pal is essential. In this work, we characterized OmpA2, an OmpAlike protein, in this bacterium. Our results showed that this protein is required for normal stalk growth and that it plays a minor role in the stability of the outer membrane. An OmpA2 fluorescent fusion protein showed that the concentration of this protein decreases from the stalk to the new pole. This localization pattern is important for its function, and it depends on the position of the gene locus in the chromosome and, as a consequence, in the cell. This result suggests that little diffusion occurs from the moment that the gene is transcribed until the mature protein attaches to the cell wall in the periplasm. This mechanism reveals the integration of different levels of information from protein function down to genome arrangement that allows the cell to self-organize.

Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms

“…Both OmpA2 structural domains are necessary for gradient formation. In our previous work, we showed that in C. crescentus, OmpA2 forms a concentration gradient from the early stages of the cell cycle and it becomes stronger as the cell grows and ages (17). To evaluate the contribution of each of the OmpA2 structural domains to the formation of the gradient, we substituted the wild-type ompA2 allele with truncated versions that consisted of only the ␤-barrel or the C-terminal PBD of OmpA2 fused to the mCherry (mCh) fluorescent protein ( Fig.…”

“…In a previous work, we proposed that the OmpA2 polar gradient forms before the protein is immobilized by its interaction with the cell wall or by its insertion in the OM (17). FRAP experiments showed that the full OmpA2 protein does not diffuse, and in consequence, the gradient must form before the protein is anchored.…”

“…Protein gradients in bacteria are rare but they play an important role in their organization (16). In C. crescentus, the presence of OmpA2 and the orientation of its concentration gradient are relevant for the cell physiology and for the synthesis of the stalk (17). Although OMPs are a major component of the OM, there is scarce information about their localization and mobility (18).…”

“…For Western blots, samples were mixed with sample buffer (final concentrations of 50 mM Tris [pH 6.8], 1% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 2 mM EDTA) and incubated for 15 min at 45, 60, 80, or 100°C for the mobility shift assays or at 100°C for regular Western blots. SDS-PAGE resolved proteins were transferred to a 0.45-m nitrocellulose membrane, incubated for ϳ4 h with a mouse red fluorescent protein (mRFP) polyclonal anti-RFP antibody raised against 6ϫHis-tagged mRFP (1:20,000 vol/vol) (17). For detection, an alkaline phosphatase conjugated to an anti-mouse antibody (1:30,000 vol/vol; Sigma) was used together with the CDP-Star/Nitro-block substrate (Thermo Fisher Scientific).…”

A New Essential Cell Division Protein in Caulobacter crescentus

“…Plasmid sequences were confirmed and the genotypes of the strains were verified by PCR and phenotype or only by phenotype when the strain was obtained from a transduction. For Western blots, SDS-PAGE-resolved proteins were transferred to nitrocellulose membranes and were incubated with a mouse polyclonal anti-red fluorescent protein (anti-RFP) antibody raised against 6ϫHis-tagged mouse RFP (65) or with a commercial anti-GFP monoclonal mouse antibody (Clontech). For detection, an alkaline phosphatase-conjugated anti-mouse antibody (Sigma) was used together with Tropix CDP-Star/Nitro-block substrate.…”