In this paper, we describe the construction and characterization of a null allele of the hfq gene in the bacterium S. oneidensis. Loss of the hfq gene produces an assortment of phenotypes, each of which is fully complemented by an exogenously supplied copy of the wild type hfq gene. To our knowledge, this is the first report of an hfq gene knockout in a dissimilatory metal reducing bacterium. Given the varied roles played by Hfq in diverse bacteria, we expect that this mutant will be both a useful tool for analyzing sRNA function in S. oneidensis as well as for understanding Hfq function in general.
It is clear from our analyses that S. oneidensis Hfq positively regulates exponential phase growth. The exponential phase growth defect of the hfq mutant is not growth medium specific, as we observe slow exponential phase growth in both complex and defined media. In addition, we observe this defect when cells are grown under both aerobic and anaerobic conditions. It is not yet clear why the hfq mutant grows slowly when nutrients are plentiful. It is possible that the hfq mutant growth phenotype is a result of a defect in nutrient acquisition, a possibility suggested by the fact that hfq mutants in a variety of bacteria express lower levels of genes involved in nutrient uptake
[6, 24–26]. It is also possible that the hfq mutant has more general set of metabolic defects that underlie its slow growth phenotype, which may explain why the hfq mutant is less efficient in Cr(VI) reduction. Alternatively, hfq may have a more specific role in utilization of Cr(VI) as a terminal electron acceptor.
A second notable hfq mutant growth phenotype is the failure of mutant cultures to achieve a terminal cell density as high as those seen in wild type cultures. Though it is not yet clear what underlies this mutant phenotype, it is possible that the hfq mutant is unable to fully utilize the available nutrients in the medium or that it exhausts a nutrient that is rate limiting for growth more rapidly than wild type cells. Alternatively, the hfq mutant may produce more of, or be more sensitive to, at least one growth-suppressing product produced during S. oneidensis growth.
Strikingly, S. oneidensis hfq mutant cultures exhibit a severe loss of colony forming units in stationary phase, with cultures often displaying no detectable CFU. One possibility is that Hfq promotes cell survival in stationary phase, and thus loss of hfq results in loss of culture viability. An alternative explanation is that Hfq functions to prevent cells from entering a viable but not culturable (VBNC) state
, and thus reduced CFU/ml counts in hfq∆ mutant cultures are due to hfq∆ cells precociously assuming VBNC status. Both of these models are supported by the fact that moderate overexpression of Hfq results in higher CFU/ml counts during stationary phase when compared to cells with wild type Hfq protein levels. Further experimentation will be required to differentiate between these two possible explanations for the greatly reduced CFU/ml counts in hfq∆ stationary phase cultures.
Because the hfq mutant is highly sensitive to oxidative stress, it is possible that the stationary phase survival defect in hfq mutant cells is a consequence of poor resistance to oxidative stress. Multiple Hfq-dependent sRNAs (arcZ, dsrA, and rprA) positively regulate expression of the stationary phase sigma factor RpoS in other systems
[28–30]. Thus, it is possible that loss of Hfq in S. oneidensis causes low rpoS expression, resulting in poor induction of the rpoS regulon. Lower rpoS regulon induction may increase the oxidative stress sensitivity of the hfq mutant and consequently reduce stationary phase survival. Another possibility that remains to be explored is whether the hfq mutant’s sensitivity to oxidative stress is due to altered function of superoxide dismutase (sodB – So_2881) and/or one or more of the four genes predicted to encode proteins with catalase activity katB (So_1070), So_1771.2, katG2 (So_4405), and katG1 (So_0725)]
. Finally, it will be of interest to determine whether S. oneidensis contains an hfq-dependent OxyR-OxyS system that is involved in response to oxidative stress as in other systems
We are currently investigating the mechanisms by which S. oneidensis Hfq promotes growth, terminal culture density, and stationary phase survival. However, given that Hfq has been broadly implicated in the function of many sRNAs in other systems
, the S. oneidensis hfq mutant generated in this study will facilitate analysis of the roles of Hfq and sRNAs in adaptation to a wide range of environmental conditions. This is of particular interest since a previous study demonstrated that S. oneidensis sRNAs do not always have completely overlapping functions with their homologs in other systems