Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference

Abstract

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Developing rationally-designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii and we developed a CRISPR-interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC-family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. Importance New approaches are urgently needed to control A. baumannii, one of the most drug resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene-regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

Interplay between the cell envelope and mobile genetic elements shapes gene flow in populations of the nosocomial pathogen Klebsiella pneumoniae

“…Accordingly, Δ wza and Δ wzy mutants, but not Δ wcaJ , lead to defects in the cell envelope of the strain Kpn SGH10 [ 51 ]. In Acinetobacter baumannii , high-density transposon mutagenesis also recently revealed that inactivation of genes involved in the last steps of capsule biosynthesis is much more deleterious than those encoding the early steps [ 52 ].…”

A new class of cell wall-recycling L,D-carboxypeptidase determines β-lactam susceptibility and morphogenesis in Acinetobacter baumannii

“…We used CRISPRi (25) to validate the negative genetic interactions between elsL and the strongest hits within each pathway ( ldt Ab , RS13190-zapA, blhA , and mlaC-F ). Chimeric single guide RNAs (sgRNAs) were designed to target the 5’ end of each locus (Fig.…”

“…CRISPRi allows efficient knockdown of operons (26); RS13190-zapA and mlaC-F are therefore each likely to be co-repressed in this strategy. The effect of knockdown on colony growth was compared with two parallel controls—(1) growth in the absence of dCas9 inducer (-aTc), and (2) growth with a control, non-targeting sgRNA (25). CRISPRi of each locus in the WT strain resulted in colony growth that was at or near control levels (Fig.…”

“…sgRNAs were constructed by PCR-amplifying 24-base, PAM-adjacent target regions satisfying previously described criteria (57, 58), followed by cloning directly into the SpeI and ApaI sites of pYDE007 (25) and verification by restriction digestion and sequencing. Δ elsL and Δ ldt Ab strains with inducible dcas9 were isolated by introducing pYDE009 (25) into EGA738 and EGA739 via four-parental mating (55, 59); transposants were isolated on VBM-Gm10 plates, generating YDA186 and YDA095, respectively. Integration at the attTn7 locus was confirmed by PCR and loss of pYDE009 was confirmed by screening on Cb plates (60, 61).…”

“…Sequencing reads were quality-filtered, clipped of adapters, and mapped to the A. baumannii chromosome (NZ_CP012004) with Bowtie (25). Mapped reads were tabulated in wig format according to the position of their TA sites in the NZ_CP012004 genome using custom python scripts (25).…”