Two selection protocols were used to screen a C. acetobutylicum genomic library for preferential growth in the presence of butanol, with protocol II providing superior outcomes. Monitoring the progression of enrichment over serial challenges generated greater genetic detail of the enrichment process.
Among the many identified candidate genes imparting solvent tolerance, we examined the impact of two genes: CAC0003 and CAC1869. The improvement in solvent tolerance was markedly better for 824(pCAC1869) than for 824(pCAC0003). When the MIC of actively growing cultures was measured (Fig. ), both 824(pCAC1869) and 824(pCAC0003) continued to grow in medium containing as much as 1.4% (vol/vol) butanol, while the 824(pIMP1) control strain was not similarly capable. For comparison, the 80 to 90% improvement in solvent tolerance for strain 824(pCAC1869) is equivalent to the 85% improvement in solvent tolerance found for strain 824(pGROE1) (50
The transcriptional patterns of the 16 enriched genes that are also the start of a predicted TU were examined using data from two DNA microarray transcriptional analyses from our laboratory (2
). One study detailed the transcriptional pattern of exponential- and transitional-phase events (3
), while another detailed the transcriptional response of exponential-phase C. acetobutylicum
cultures to a single bolus of 0.46% (vol/vol) butanol (2
). Of the 16 enriched genomic inserts, CAC1869, CAC3435, and CAC3359 were maximally transcribed at the onset of the transitional phase. Conversely, CAC1463 and CAC1874 were transcribed predominantly in the exponential phase, while CAC0003, CAC3143, CAC0854, and CAC0395 were all downregulated at the onset of the transitional phase (3
). Interestingly, transcription of these 16 enriched genes was not significantly affected by butanol stress (2
). We conclude that a stress response is not necessarily a good method for identifying genes imparting tolerance to the stressor.
CAC1869 is annotated as a singleton transcriptional regulator, and for several reasons it is reasonable to assign a role in the regulation of transitional-phase events. CAC1869 is maximally transcribed just prior to the induction of the solventogenic genes aad
, and ctfB
and remains actively transcribed through the transitional phase (3
). Also, protocol II favored inserts that delay transitional-phase phenomena, making cells that harbor them better able to continue vegetative growth upon inoculation into the next challenge flask. Consistent with that, 824(pCAC1869) exhibited prolonged metabolic activity through the transitional phase compared to that of the 824(pIMP1) control strain.
Transcriptional regulators are important for tolerant phenotypes (1
). CAC1869 has homology to the xenobiotic-responsive element (XRE) family of regulatory proteins (35
). Twenty-four other XRE-like regulatory proteins reside in the C. acetobutylicum
). CAC1869 shows the closest homology to the Bacillus cereus
NVH 391-98 gene bcer98draft_2450 (34% identity), but no similar homolog is present in the other functionally annotated clostridia (Clostridium perfringens
and Clostridium tetani
). XRE-like proteins have been linked to a wide variety of cellular functions (9
) and tolerant phenotypes (6
). The protein product of la867 is critical for acid tolerance in Lactobacillus acidophilus
) and has homology to gadR
, a positive regulator of the Lactococcus lactis
acid resistance operon gadBC
). One of the primary genes for regulating the recovery of Deinococcus radiodurans
from ionizing radiation is the XRE-like regulator dr2574. Finally, SinR is an XRE-like regulator that forms a tetramer that binds promoter regions and represses the transcription of genes (e.g., spo0A
) that are necessary for the initiation of the sporulation cascade (17
). In the case of CAC1869, further study is necessary in order to determine which genes are directly regulated as well as the precise regulatory mechanism for tolerance imparted by CAC1869 overexpression.
With both library selection protocols, mixed-cell populations exhibited growth in media containing 1.56% butanol. However, neither 824(pIMP1) nor 824(pCAC0003) nor 824(pCAC1869) was able to grow when inoculated in medium containing more than 1.3% butanol (Fig. ), and actively growing cultures were completely inhibited at concentrations exceeding 1.6% butanol (Fig. ). This suggests that library-bearing cultures with a mixture of inserts reproducibly tolerated higher butanol levels than cultures with only a single insert or the pIMP1 control. Two explanations are that this is either an inherent trait of the mixed population or the result of unidentified chromosomal events leading to improved solvent tolerance. The 824(pIMP1) control strain, when subjected to stationary-phase challenges with increasing butanol concentrations, apparently did not display a similar chromosomal event, surviving at only up to 1.3% butanol (transfer 10) (see Table S2 in the supplemental material) and indicating either a low frequency of occurrence or a role for library inserts in producing these beneficial chromosomal modifications.
The identification of additional solvent tolerance elements is possible for several reasons. First, the pLib1 library provided coverage for approximately 73% of the C. acetobutylicum
genome. Second, the lack of a constitutive promoter on the pIMP1 vector precluded the expression of library inserts separated from their natural promoter. Finally, the preferential uptake of plasmids containing smaller library inserts limited the sizes of genes and operons screened in protocols I and II. This likely explains why previously identified tolerance elements, such as the groESL
operon (~2.2 kb) (50
), were not identified in this study.
Despite these shortcomings of the pLib1 genomic library, the method and results presented here should prove useful for future efforts to define the genetic basis for complex phenotypes such as tolerance to various toxic chemicals. Visualizing the dynamic process of library insert enrichment with DNA microarrays provides greater genetic detail than previous efforts that aimed to characterize the result of the enrichment process (18
). Enriched genes identified in this study require further characterization of their role in solvent tolerance through a combination of constitutive gene overexpression and DNA microarray transcriptional profiling. In addition, co-overexpression of multiple enriched genes in a single culture may provide additional tolerance above that imparted singly.