Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10 to 10 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-β-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.
Despite the urgent need for sustained development of novel antibacterial compounds to combat the drastic rise in antibiotic resistant and emerging bacterial infections, only a few clinically relevant antibacterial drugs have been recently developed. One of the bottlenecks impeding the development of novel antibacterial compounds is the identification of new enzymatic targets. The nutritionally essential amino acid anabolic pathways, for example lysine biosynthesis, provide an opportunity to explore the development of antibacterial compounds, since human genomes do not possess the genes necessary to synthesize these amino acids de novo. The diaminopimelate (DAP)/lysine (lys) anabolic pathways are attractive targets for antibacterial development since the penultimate lys precursor meso-DAP (m-DAP) is a cross-linking amino acid in the peptidoglycan (PG) cell wall of most Gram-negative bacteria and lys plays a similar role in the PG of most Gram-positive bacteria, in addition to its role as one of the 20 proteogenic amino acids. The L,L-diaminopimelate aminotransferase (DapL) pathway was recently identified as a novel variant of the DAP/lys anabolic pathways. The DapL pathway has been identified in the pathogenic bacteria belonging to the genus; Chlamydia, Leptospira, and Treponema. The dapL gene has been identified in the genomes of 381 or approximately 13% of the 2771 bacteria that have been sequenced, annotated and reposited in the NCBI database, as of May 23, 2014. The narrow distribution of the DapL pathway in the bacterial domain provides an opportunity for the development and or discovery of narrow spectrum antibacterial compounds.
Shrub willow, Salix spp. and hybrids, is an important bioenergy crop. Here we report the whole-genome sequences and annotation of 13 endophytic bacteria from stem tissues of Salix purpurea grown in nature and from commercial cultivars and Salix viminalis × Salix miyabeana grown in bioenergy fields in Geneva, New York.
L,L-Diaminopimelate aminotransferase (DapL) is an enzyme required for the biosynthesis of meso-diaminopimelate (m-DAP) and L-lysine (Lys) in some bacteria and photosynthetic organisms. m-DAP and Lys are both involved in the synthesis of peptidoglycan (PG) and protein synthesis. DapL is found in specific eubacterial and archaeal lineages, in particular in several groups of pathogenic bacteria such as Leptospira interrogans (LiDapL), the soil/water bacterium Verrucomicrobium spinosum (VsDapL) and the alga Chlamydomonas reinhardtii (CrDapL). Here we present the first comprehensive inhibition study comparing the kinetic activity of DapL orthologs using previously active small molecule inhibitors formerly identified in a screen with the DapL of Arabidopsis thaliana (AtDapL), a flowering plant. Each inhibitor is derived from one of four classes with different central structural moieties: a hydrazide, a rhodanine, a barbiturate, or a thiobarbituate functionality. The results show that all five compounds tested were effective at inhibiting the DapL orthologs. LiDapL and AtDapL showed similar patterns of inhibition across the inhibitor series, whereas the VsDapL and CrDapL inhibition patterns were different from that of LiDapL and AtDapL. CrDapL was found to be insensitive to the hydrazide (IC₅₀ >200 μM). VsDapL was found to be the most sensitive to the barbiturate and thiobarbiturate containing inhibitors (IC₅₀ ∼5 μM). Taken together, the data shows that the homologs have differing sensitivities to the inhibitors with IC₅₀ values ranging from 4.7 to 250 μM. In an attempt to understand the basis for these differences the four enzymes were modeled based on the known structure of AtDapL. Overall, it was found that the enzyme active sites were conserved, although the second shell of residues close to the active site were not. We conclude from this that the altered binding patterns seen in the inhibition studies may be a consequence of the inhibitors forming additional interactions with residues proximal to the active site, or that the inhibitors may not act by binding to the active site. Compounds that are specific for DapL could be potential biocides (antibiotic, herbicide or algaecide) that are nontoxic to animals since animals do not contain the enzymes necessary for PG or Lys synthesis. This study provides important information to expand our current understanding of the structure/activity relationship of DapL and putative inhibitors that are potentially useful for the design and or discovery of novel biocides.
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