title: The waaL gene mutation compromised the inhabitation of Enterobacter sp. Ag1 in the mosquito gut environment.
abstract: BACKGROUND
  The mosquito gut harbors a variety of bacteria that are dynamically associated with mosquitoes in various contexts. However, little is known about bacterial factors that affect bacterial inhabitation in the gut microbial community. Enterobacter sp. Ag1 is a predominant Gram negative bacterium in the mosquito midgut.
  
  
  METHODS
  In a mutant library that was generated using transposon Tn5-mediated mutagenesis, a mutant was identified, in which the gene waaL was disrupted by the Tn5 insertion. The waaL encodes O antigen ligase, which is required for the attachment of O antigen to the outer core oligosaccharide of the lipopolysaccharide (LPS).
  
  
  RESULTS
  The waaL(-) mutation caused the O antigen repeat missing in the LPS. The normal LPS structure was restored when the mutant was complemented with a plasmid containing waaL gene. The waaL(-) mutation did not affect bacterial proliferation in LB culture, the mutant cells grew at a rate the same as the wildtype (wt) cells. However, when waaL(-) strain were co-cultured with the wt strain or complemented strain, the mutant cells proliferated with a slower rate, indicating that the mutants were less competitive than wt cells in a community setting. Similarly, in a co-feeding assay, when fluorescently tagged wt strain and waaL(-) strain were orally co-introduced into the gut of Anopheles stephensi mosquitoes, the mutant cells were less prevalent in both sugar-fed and blood-fed guts. The data suggest that the mutation compromised the bacterial inhabitation in the gut community. Besides, the mutant was more sensitive to oxidative stress, demonstrated by lower survival rate upon exposure to 20 mM H₂O₂.
  
  
  CONCLUSIONS
  Lack of the O antigen structure in LPS of Enterobacter compromised the effective growth in co-culture and co-feeding assays. In addition, O-antigen was involved in protection against oxidative stress. The findings suggest that intact LPS is crucial for the bacteria to steadily stay in the gut microbial community.
authors: Biology Department, New Mexico State University, Las Cruces, NM, 88003, USA. dpei@nmsu.edu.
authors: Dong
authors: D
authors: Pei
authors: Biology Department, New Mexico State University, Las Cruces, NM, 88003, USA. jinjiang@nmsu.edu.
authors: Jinjin
authors: J
authors: Jiang
authors: Biology Department, New Mexico State University, Las Cruces, NM, 88003, USA. ivyyu@nmsu.edu.
authors: Wanqin
authors: W
authors: Yu
authors: Entomology Department, Cornell University, Isaac, NY, USA. pk476@cornell.edu.
authors: Phanidhar
authors: P
authors: Kukutla
authors: Biology Department, New Mexico State University, Las Cruces, NM, 88003, USA. hondrouentillie64@gmail.com.
authors: Alejandro
authors: A
authors: Uentillie
authors: Biology Department, New Mexico State University, Las Cruces, NM, 88003, USA. jxu@nmsu.edu.
authors: Jiannong
authors: J
authors: Xu
mesh_terms: D000818:Animals; D000852:Anopheles / Q000382:microbiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics; D004754:Enterobacter / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016296:Mutagenesis; D009154:Mutation; D018384:Oxidative Stress
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D006861:Hydrogen Peroxide