Symbiotic nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which it enters the plant root. Once these invasion structures reach the target cells in the interior of the plant root, the bacteria are endocytosed within a host cell membrane-derived compartment. In the microaerobic environment provided by the host cell, the bacteria differentiate into a specialized form called a bacteroid. The bacteroid form expresses the oxygen-sensitive enzyme nitrogenase that catalyzes the conversion of atmospheric nitrogen to ammonia. The dissection of the bacterial and plant signalling pathways that are involved in each stage of the invasion process has been facilitated by the complete genomic sequencing of Sinorhizobium meliloti and the near complete sequencing of the genome of the model host plant Medicago truncatula. Rhizobial bacteria interact very differently with the plant innate immune system than other groups of bacteria. Rhizobia lack some of the microbial molecular patterns that provoke plant defence responses. Additionally, legume plants differ from other plant families in that they lack the ability to perceive and respond defenceively to other microbial molecular patterns. Symbiotic rhizobial bacteria are similar to pathogenic bacteria such as Brucella spp, in that they both form chronic infections of eukaryotic cells within a host-derived membrane compartment, and require some of the same bacterial factors for survival within the host. These factors include the correct structure of the lipopolysaccharide core and lipid A, presence of cyclic β-glucans, and a common bacterial regulatory circuitry.

Inducing immune bee genes: Honey bees are prone to parasitism by the Varroa mite, which is a vector for several bee pathogens. However, honey bees are also host to the symbiotic gut bacterium Snodgrassella alvi. Leonard et al. engineered S. alvi to produce double-stranded RNA (dsRNA)—a stimulus for insect RNA interference defense responses—from a plasmid containing two inverted promoters tagged with a fluorescent label (see the Perspective by Paxton). This dsRNA module can be targeted to interfere with specific bee genes as well as crucial viral and mite genes. The authors found that gene expression could be blocked for at least 15 days as the symbionts established in the bees' guts and continuously expressed the dsRNA constructs. S. alvi with specifically targeted plasmids not only suppressed infection with deformed wing virus but also effectively reduced Varroa mite survival. Science , this issue p. 573 ; see also p. 504

Rates of infection with hospital-acquired Acinetobacter baumannii have exploded over the past decade due to our inability to limit persistence and effectively treat disease. A. baumannii quickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings. With depleted antibiotic options, new methods to treat A. baumannii infections are desperately needed. A comprehensive understanding detailing A. baumannii cellular factors that contribute to its resiliency at genetic and mechanistic levels is vital to the development of new treatment options. Tools to rapidly dissect the A. baumannii genome will facilitate this goal by quickly advancing our understanding of A. baumannii gene-phenotype relationships. We describe here a recombination-mediated genetic engineering (recombineering) system for targeted genome editing of A. baumannii . We have demonstrated that this system can perform directed mutagenesis on wide-ranging genes and operons and is functional in various strains of A. baumannii , indicating its broad application. We utilized this system to investigate key gene-phenotype relationships in A. baumannii biology important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance mechanisms, and biofilm formation. In addition, we have demonstrated that both the formation and movement of type IV pili play an important role in A. baumannii biofilm. IMPORTANCE Acinetobacter baumannii is the causative agent of hospital-acquired infections, including pneumonia and serious blood and wound infections. A. baumannii is an emerging pathogen and has become a threat to public health because it quickly develops antibiotic resistance, making treatment difficult or impossible. While the threat of A. baumannii is well recognized, our understanding of even its most basic biology lags behind. Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques. Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products. This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships. To demonstrate the power of recombineering in dissecting A. baumannii biology, we use this system to establish key gene-phenotype relationships important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance, and biofilm formation.

Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii , fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella , increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxL _Ab ( A. baumannii LpxL) and LpxM _Ab ( A. baumannii LpxM), which transfer one and two lauroyl (C _12:0 ) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxM _Ab -dependent acylation of lipid A is essential for A. baumannii desiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxM _Ab -dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections. IMPORTANCE Acinetobacter baumannii infections can be life threatening, and disease can progress in a variety of host tissues. Current antibiotic regimen and disinfectant strategies have failed to limit nosocomial A. baumannii infections. Instead, the rate of A. baumannii infection among health care communities has skyrocketed due to the bacterium's adaptability. Its aptitude for survival over extended periods on inanimate objects, such as catheters, respirators, and surfaces in intensive care units, or on the hands of health care workers and its ability to rapidly develop antibiotic resistance make A. baumannii a threat to health care communities. Emergence of multidrug- and extremely drug-resistant A. baumannii illustrates the ineffectiveness of current prevention and treatment options. Our analysis to understand how A. baumannii resists cationic antimicrobial peptide (CAMP)-mediated and desiccative killing revealed two lipid A acyltransferases that produce protective hepta-acylated lipid A. Our work suggests that inhibiting lipid A biosynthesis by targeting the acyltransferase LpxM _Ab ( A. baumannii LpxM) could provide a novel target to combat this pathogen.