Molecular characterization of arginine deiminase pathway and re-annotation of the laribacter hongkongensis genome

Abstract

Laribacter hongkongensis, a recently discovered Gram-negative bacillus, is associated with fish-borne community-acquired gastroenteritis and traveler’s diarrhea. Although its complete genome was sequenced, getting more accurate genome information by reannotation would be optimal for analyzing and performing further studies. Similar to other gastrointestinal tract pathogens, L. hongkongensis has to overcome the hostile acidic condition in the stomach when they transmit through the oral route. Exploring the molecular mechanisms of L. hongkongensis for resisting acidic condition would be essential for us to understand its lifecycle, the transition from environments to humans and pathogenesis.

A comprehensive re-annotation on the genome of L. hongkongensis was performed, using systematic method which combines composition- and similarity-based approaches. Eighty-six hypothetical genes were eliminated from the previous annotation by composition-based method and eight new genes were picked up by a combination of the ab initio gene finder and similarity alignment, which were further verified by RT-PCR. Based on similarity search, functional information of some hypothetical proteins was updated. The re-annotation on the genome of L. hongkongensis provides more accurate information of genome sequence, which would benefit for the investigation on our bacterium in its metabolism, environmental adaptation and pathogenesis.

Using the re-annotation results, potential acid resistance systems (urease system, ADI system encoded by two adjacent arc gene cassettes) were observed by similarity alignment. Different mutants on these two systems were constructed and subjected to in vitro acid resistance, intracellular survival and animal studies. All of the urease-negative mutants exhibited no urease activity and similar survival under acidic condition (pH < 3), which was slightly decreased when compared to wild type HLHK9. Both of the arc gene cassettes contributed to ADI activity and only the survival of double mutant HLHK9ΔarcA1/arcA2 (pH < 3) dramatically decreased when compared to that of wild type HLHK9. For the intracellular survival in macrophages, HLHK9ΔarcA1/arcA2 and HLHK9ΔureA/arcA1/arcA2 were dramatically dropped but HLHK9ΔureA was slightly reduced, compared to HLHK9. The transcription level of arcA1, arcA2 and ureA genes were all increased. HLHK9ΔureA exhibited similar survival to that of HLHK9 after oral injection to mice, but HLHK9ΔarcA1/arcA2 and HLHK9ΔureA/arcA1/arcA2 were significantly decreased. These data supported that the ADI pathway plays a much more crucial role for resisting acidity and intracellular survival in L. hongkongensis, instead of urease. The regulatory mechanisms of these two adjacent arc gene cassettes by environmental stresses and transcriptional factors were studied. We showed that these two arc gene cassettes are transcribed separately and displayed distinct outcomes under multiple environmental stresses (acidity, temperature, oxygen and arginine supplement). Moreover, a putative transcriptional regulator ArgR was identified and proved to mediate the arginine regulation on ADI pathway genes via interacting with binding sites located on the promoter regions. Disruption of argR significantly reduced the bacterial survival in macrophages and the expression of argR gene 8 h post-infection was upregulated. Finally, we demonstrated that environmental stresses and transcriptional regulator ArgR also regulate the gene expression from arginine biosynthesis pathways, indicating the importance of arginine metabolism as an adaptive mechanism in sensing and responding to environmental stresses.