Genetic determinants of swimming motility in the squid light-organ symbiont Vibrio fischeri
The evolutionary relationship among Vibrio fischeri isolates bobtail squid, Euprymna scolopes, in particular, on Oahu, Hawaii (Wollenberg and Ruby, ). We are also grateful for the statistical advice of C Ané and. The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting fischeri lives in a cooperative association with the Hawaiian bobtail squid, Euprymna scolopes. The relationship between V. fischeri and its squid host is complex and highly specific. .. Advice on statistical treatments was provided by A. Taylor. Hawaiian bobtail squid (Euprymna scolopes UNH microbiologists have Robot Therapy which plays host to the light-producing bacterium Vibrio fischeri. symbiotic relationship between bobtail squid and V. fischeri in this.
The ciliated appendages regress within days of inoculation. Through observations of pycnotic nuclei, indicators of apoptosis programmed cell deathit was determined that the appendages are diminished by massive cell death. When treated with antibiotics at various time points, it was discovered that the bacteria only need to be present for at most 8 hours to initiate the apoptosis program, implying that the bacteria provide a transient and irreversible signal to the host cells.
It is also significant to note that the bacteria do not stay in contact with the tissue that regresses. Utilizations of mass spectrometry and fractionation revealed that the bacterial signal is tracheal cytotoxin TCTa disaccharide-tetrapeptide monomer of the bacterial surface molecule peptidoglycan. At first, it was thought that the cell death was triggered by lipopolysaccharide LPS and peptidoglycan PGN working together in some concentration but experimentation demonstrated that this combination was not consistent and did not elicit the same level of apoptosis.
Koropatnick, This TCT generated cell death first causes the elongated ciliated ridge to regress and be replaced with non-ciliated cells. The number of dying cells within the light organ peaks at 14 hours post-inoculation but cells continue to die for the first 5 days. Within 3 days in unfiltered seawater, the posterior appendage is completely absent and the anterior appendage is markedly reduced in size.
In sterile seawater, and still infected, the appendages are only slightly decreased in size. Montgomery TCT provokes an accumulation of blood cells hemocytes in the ciliated fields that act like macrophages, digesting the soon- to- be dying epithelial cells.
Koropatnick, It is not certain that TCT specifically elicits the rest of the morphology changes though they are highly correlated with the presence of the bacteria. Through western blotting, this large-scale cell death activated by TCT has been shown to utilize p53 signaling between cells. P53 signaling is a known tumor suppressor gene. In the absence of a stimulating signal, p53 forms a complex with MDM2 in the nucleus, causing a move into the cytoplasm, where it gets ubiquinated and degraded.
When the proper signal does come along, p53 is stabilized through phosphorylation, blocking MDM2 binding. The p53 protein then is able to build up in the nucleus and performs its role as a transcription factor, leading to apoptosis. The western blot of aposymbiotic organisms showed a higher concentration of p53 in the cytoplasm whereas organisms infected with V.
NO production is typically an immunological response against MAMPs microbe associated molecular patterns. This interaction with the immune system is extremely interesting and opens up a whole new realm of research that could be conducted concerning this symbiotic relationship.
Another major change associated with the presence of the Vibrio fischeri is the extensive branching of the epithelial crypts. There are still 3 separate crypts but it is harder to distinguish due to the branching.
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The branching also causes the modification of the ducts connecting each crypt to their separate pores. Now, the crypts connect to one 5-branched duct, which leads to the surface to one pore.
There is one branch to each crypt, one branch to the anterior yellow filter, and one branch to the posterior yellow filter. In aposymbiotic squid, branching still occurs but it is much slower and not as elaborate. Montgomery, The individual duct cells are also altered with the bacterial colonization. At this point, these cells contain a large number of cytoplasmic vesicles, hypothesized to have a secretory role in the daily expulsion of the Vibrio fischeri cells.
In aposymbiotic duct cells, there are no inclusions and the cells are more heterogenous in appearance. In addition, the crypts are wrapped in dynamic diverticula, which are blind tubes derived from the ink sac described earlier. Photo courtesy of thechelseascrolls. Photo courtesy of biology. While the oxygen is unnecessary for sustaining A. Squid detect light levels through the use of multiple organs situated on the top of the animal and can adjust the light levels within minutes.
Having a culture of A.
The light organ of a newly hatched squid. Aliivibrio fischeri enters the light organ though pores on either side of the organ. These can be seen as the three small holes on the left and right side of the image. The blue color is produced by the bacteria. In these bacteria, additional proteins are important for motility, including the regulators FlhF and FlhG, which control flagellar number in V. In the work described here, we sought to understand the genetic basis of flagellar motility, an essential cellular behavior for the host-associated lifestyle of V.
We performed both forward and reverse genetic analyses, coupled with transcriptional profiling, to identify the genes that contribute to normal motility during symbiosis. Experimental Procedures Bacterial strains and media Strains and plasmids used in this work are listed in Table S1. When appropriate, antibiotics were added to media at the following concentrations: Growth media were solidified with 1. Construction and motility screening of an arrayed transposon mutant collection To investigate the genetic basis of flagellar motility, we conducted random mutagenesis with pMJM10, a conjugatable plasmid that encodes an erythromycin-resistant transposon.
Construction from pEVS was accomplished as follows. The resulting plasmid was named pMJM8 contains a single outward-facing T7 promoter at the upstream end of the transposon, relative to the orientation of the erm cassette.McFall-Ngai (U. Hawaii Manoa) 1: Living together: The symbiosis of host-microbial interactions
Second, vector sequence adjacent to the transposon upstream end was modified to introduce MseI and TspI sites by site-directed mutagenesis. The construction of the MB mutant collection is illustrated in detail in Figure S1. Briefly, we mutagenized V. We screened the arrayed mutants for strains that contained a transposon insertion erythromycin resistantbut that additionally did not retain the donor plasmid kanamycin sensitive.
These strains were rearrayed, frozen as glycerol stocks, and saved for further analysis. The final library contains 23, mutant strains and is termed the MB Collection.
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This collection contains only trays that, upon screening for motility phenotypes, provided reproducible phenotypes across the entire agar tray during the motility screen. We observed a correlation between aberrant motility screen results and subsequent inability to regrow strains from plate freezer stocks.
We attributed such results to harsh growth conditions during passage of the strains e. In this manner, motility screening provided a quality control step for the entire collection. Trays that did not pass this step were not included in the final collection or in the screen results. Cultures were then normalized to an OD of 0.