Genomics of Acinetobacter baumannii iron uptake

Abstract

Iron is essential for growth in most bacteria due to its redox activity and its role in essential metabolic reactions; it is a cofactor for many bacterial enzymes. The bacterium Acinetobacter baumannii is a multidrug-resistant nosocomial pathogen. A. baumannii responds to low iron availability imposed by the host through the exploitation of multiple iron-acquisition strategies, which are likely to deliver iron to the cell under a variety of environmental conditions, including human and animal infection. To date, six different gene clusters for active iron uptake have been described in A. baumannii , encoding protein systems involved in (i) ferrous iron uptake (feo); (ii) haem uptake (hemT and hemO); and (iii) synthesis and transport of the baumannoferrin(s) (bfn), acinetobactin (bas/bau) and fimsbactin(s) (fbs) siderophores. Here we describe the structure, distribution and phylogeny of iron-uptake gene clusters among >1000 genotypically diverse A. baumannii isolates, showing that feo, hemT, bfn and bas/bau clusters are very prevalent across the dataset, whereas the additional haem-uptake system hemO is only present in a portion of the dataset and the fbs gene cluster is very rare. Since the expression of multiple iron-uptake clusters can be linked to virulence, the presence of the additional haem-uptake system hemO may have contributed to the success of some A. baumannii clones.

Keywords: Acinetobacter baumannii; haem; iron uptake; phylogeny; sequence type; siderophore.

Fig. 1.

Genetic structure of iron-uptake gene clusters in A. baumannii . Gene annotations and positions refer to the genome of A. baumannii ACICU (NZ_CP031380.1), except for the fimsbactin(s) gene cluster, which refers to A. baumannii ATCC 17978 (NZ_CP012004.1). Different colours denote different gene functions. Genes with unknown function are shown in white. hp, hypothetical protein.

Fig. 2.

Distribution of iron-uptake gene clusters among 1071 A . baumannii isolates. (a) The colour code designates each iron-uptake cluster combination; black and white squares denote the presence or absence of the iron-uptake cluster, respectively. The number of positive isolates for each combination is provided. The pie chart on the right shows the relative abundance of each combination according to their colour codes. (b) Sequence type distribution of the two most frequent iron-uptake gene cluster combinations, namely isolates lacking only fbs (n=717) or both fbs and hemO (n=309), relative to all combinations (n=1071).

Fig. 3.

Genetic structure of transposons carrying the fimsbactin(s) gene cluster. Comparison of transposon Tn6171 in A. baumannii strain D36 to transposons from strains ATCC 17978 (ID: 2240), E437 (ID: 6116), F447 (ID: 6117), 397 971 (ID: 2978), AB0365 (ID: 5007), G20251008 (ID: 6582) and G18255951 (ID:6577). The arrows indicate the extent and orientation of genes and ORFs, with genes belonging to the fbs cluster in orange, insertion sequences in yellow, transposons in green, and all other genes in grey. The double slash denotes an interruption between two different contigs.

Fig. 4.

Distribution of the hemO gene cluster according to the core genome-based phylogeny of 1071 A . baumannii strains. The tree was constructed from core genome SNP analysis of 1071 strains from different STs, as shown in the colour code legend. The presence of the hemO cluster in the dataset is indicated by the black ring surrounding the tree. Internal node support was determined using the Shimodaira–Hasegawa test, with values ≥80% indicated by black-filled circles. The tree is midpoint rooted, and the scale bar represents the expected number of substitutions per site.

Fig. 5.

Evolution of the feo, hemT, bfn and bas/bau iron-uptake gene clusters in A. baumannii . Neighbour-joining trees have been generated by the alignment of the concatenated sequences of (a) the feo gene cluster from 1068 A . baumannii strains; (b) the hemT gene cluster from 1013 A . baumannii strains; (c) the bfn gene cluster from 1036 A . baumannii strains; (d) the bas/bau gene cluster from 1030 A . baumannii strains. STs are differentiated according to their colour code, as indicated in the key legend. Black-filled circles at the nodes denote bootstrap values ≥80 % (1000 replicates). The trees are midpoint rooted and the scale bars represent the expected number of substitutions per site.

Fig. 6.

Evolution of the hemO clusters and genetic configuration of the hemO region in A. baumannii . (a) Neighbour-joining tree based on the alignment of concatenated sequences of the hemO gene cluster retrieved from 736 A . baumannii strains. STs are differentiated according to their colour code, as indicated in the key legend. Black-filled circles at the nodes denote bootstrap values ≥80 % (1000 replicates). The tree is midpoint rooted, and the scale bar represents the expected number of substitutions per site. The SDF branch is shown in red. (b) (top) The genomic context of the hemO gene cluster in A. baumannii ACICU. The orange-shaded region defines the boundaries of the hemO gene cluster, spanning from the ECF sigma to the hp gene (orange). The double slash denotes a region of hemO that is not shown. The gene map is not to scale. (b) (middle) Level of sequence identity of the hemO-flanking region among 732 and 729 hemO-posive genomes, including 3 upstream and 3 downstream genes, respectively. Green indicates 100 % identity, greeny-brown at least 30 % identity and red below 30 % identity. (b) (bottom) Consensus sequence of the 20 nt region between the hemO-flanking genes (DMO12_RS04680 and DMO12_RS04725, relative to the A. baumannii ACICU nomenclature) in 303 isolates lacking the hemO gene cluster, generated by the WebLogo representation (http://weblogo.threeplusone.com/ accessed January 2023). The first triplet (TTA) is the stop codon of the upstream gene (DMO12_RS04680), while the last triplet (TAA) is the stop codon of the convergent downstream gene (DMO12_RS04725). The height of the total stack indicates the degree of sequence conservation at a given position.

Fig. 7.

Evolutionary history of the hemO gene cluster in genus Acinetobacter . Unrooted neighbour-joining tree generated from the sequence alignment of the entire hemO gene cluster retrieved from 1641 genomes of different species belonging to the genus Acinetobacter . Species are differentiated by colour, as indicated in the key legend. Black-filled circles at the nodes denote bootstrap values ≥80 % (1000 replicates). The scale bar indicates the expected number of substitutions per site.