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

Abstract

BackgroundBrucella 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.ResultsWe 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.ConclusionsData 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.

BtaE, an Adhesin That Belongs to the Trimeric Autotransporter Family, Is Required for Full Virulence and Defines a Specific Adhesive Pole of Brucella suis

“…Moreover, proteins specifically recruited to the old pole or to the new pole have been reported. The PdhS histidine kinase is recruited to the old pole (19,20), and the putative DNA repair protein AidB is recruited to the new pole (18). Since both the BmaC and BtaE adhesins showed unipolar localization, the question arises as whether these adhesins are localized at a specific pole and, in that case, if they are localized at the same pole.…”

“…We also analyzed the localization of BtaE on the bacterial surface and found that it showed unipolar localization, which has not been previously reported for a trimeric autotransporter (TA). This observation, together with the previous evidence indicating that BmaC also shows unipolar localization (15), prompted us to determine whether BtaE or BmaC or both are localized at a particular pole, using markers of the new and old poles (18)(19)(20). Our observations show that BtaE and BmaC are exclusively associated with the new pole, suggesting that this pole in Brucella is specialized for adhesion.…”

“…*, significantly different from the wild type (P Ͻ 0.05). fusion proteins encoded by either pdhS-egfp (19) or aidB-yfp (18) to evaluate by immunofluorescence the relative localization of BmaC. DAPI staining and phase contrast were used to distinguish the bacterial shapes.…”

“…Brucella was transformed with a derivative of pBBR1MCS-2 (30), which carries gfp constitutively expressed (excitation at 488 nm, emission at 509 nm; filters used a bandpass [BP] range of 505 to 530 nm) (15). The strains carrying polar markers fused to fluorescent proteins were obtained as previously described by biparental conjugation with E. coli S17.1 strains carrying either pdhS-egfp (19) or aidB-yfp (18).…”

The Histidine Kinase PdhS Controls Cell Cycle Progression of the Pathogenic Alphaproteobacterium Brucella abortus

“…For the mating, yeast strains were plated on yeast extract-peptone-dextrose (YPD) medium overnight and then replicated on selective medium (i.e., depleted in uracil or histidine with 100 mM 3-aminotriazole) when appropriate, as previously described (7). For the localization screen, Gateway cloning with pools of each coding sequence (CDS) was used, as previously described for pools of multiple CDS (9).…”