The alkylation response protein AidB is localized at the new poles and constriction sites in Brucella abortus

Abstract

Background: Brucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.

Results: We were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects.

Conclusions: Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.

Figure 1

The B. abortus aidB mutant is more sensitive to EMS. The sensitivity of B. abortus wild-type, aidB mutant strain, complemented aidB mutant and aidB overexpression strains was scored by counting the c.f.u. recovered after 4 h of incubation 2YT medium at 37°C, in the presence of 0.2, 0.4 or 1% EMS. The results are expressed as the percentage of c.f.u. compared to a control in which EMS was omitted. Bacteria were obtained from cultures stopped during exponential growth phase.

Figure 2

The B. abortus AidB-YFP is localized at new poles and at constriction sites, in culture and in macrophages. The B. abortus XDB1128 strain was carrying an aidB-yfp fusion on a low copy plasmid, and pdhS-mCherry at the pdhS chromosomal locus. (A) Bacteria were grown in rich medium and the pictures were taken in exponential phase. Differential interference contrast (DIC) is shown on the left. The white arrowheads indicate the dividing cell in which two AidB-YFP foci are detectable. Each scale bar represents 2 μm. The bacterial types are schematically drawn on the right side of the pictures, as they are represented in figure 6. The two upper panels were made with non-diving bacteria, and counting was made with 125 bacteria. The two lower panels were made with dividing bacteria, and counting was made on 84 dividing bacteria. (B) RAW264.7 macrophages were infected for 2, 4, 6, or 24 h with the B. abortus strain expressing aidB-yfp (XDB1120). The infected cells were fixed and immunostained with 12G12 anti-lipopolysaccharide ("α-LPS") primary antibody and anti-mouse secondary antibody coupled to Texas Red. A majority of the bacteria present a single focus of AidB-YFP, suggesting that polar localization of this fusion is also occurring during the infection of macrophages.

Figure 3

Size distribution of B. abortus carrying AidB-YFP, in the presence or absence of an alkylating agent (EMS). The bacterial lengths were grouped in classes of 0.25 μm, and the maximum value for each class is given on the × axis. (A) Size distribution of 276 bacteria (XDB1128 strain) with AidB-YFP either at the new pole (white), the new pole and the constriction site (dark grey), or the constriction site only (black). (B) Size distribution of B. abortus (XDB1128 strain) exposed to 0.4% of EMS for 4 h (light grey, n = 340) or the unexposed control (white, n = 218, bacteria without detectable constriction). (C) DIC and fluorescence pictures of the XDB1128 strain expressing aidB-yfp and pdhS-mCherry fusions, as described in figure 2. The bacteria in the lower panels have been exposed to 0.4% EMS for 4 h in rich (2YT) medium. On the top panels, control bacteria were incubated for 4 h in 2YT in the absence of EMS. Constriction sites are indicated by arrowheads. Each scale bar represents 2 μm.

Figure 4

Growth defect of the B. abortus strain expressing the aidB-yfp fusion (XDB1120). The growth of B. abortus wild-type, aidB mutant and XDB1120 (pMR-aidB-yfp) strains was followed by recording OD at 600 nm in a Bioscreen. Duplicates (1) and (2) are shown for each strain, for 2YT (left panel) or tryptic soy broth (right panel) as culture media. In both culture media, the OD₆₀₀ during stationary culture phase of the XDB1120 strain is lower compared to the wild type control.

Figure 5

Morphological defect of the B. abortus aidB overexpressing strain. Differential interference contrast (DIC) images were taken with bacteria of the aidB (aidB⁺⁺⁺), acaD1 (acaD1⁺⁺⁺) and acaD2 (acaD2⁺⁺⁺) overexpression strains, the aidB disruption strain, and the wild-type strain with or without the control pBBR1MCS plasmid [32], without insert. Two panels are shown for the aidB overexpression strain, the only strain displaying a morphological defect during stationary culture phase. The morphological defects are multiple, with multipolar bacteria (M), Y-shaped cells (y), swollen cells (s) and some minicells (m).

Figure 6

Model for the localization of AidB-YFP along B. abortus cell cycle. The PdhS-mCherry is labelling the old pole of B. abortus. AidB-YFP is therefore localized at the new pole, as suggested by Figure 2. In dividing cells, we hypothesize that AidB-YFP is first present at the young pole (the new pole that becomes old) and at the constriction site. This localization at the young pole would be lost afterwards, allowing the generation of two sibling cells with a unique pole of AidB-YFP. The new (n), young (y) and old (o) poles are labelled. In this model, the constriction region would be the preparation site for the new poles of the sibling cells.