Adaptive gene expression in Bacillus subtilis strains deleted for tetL

Abstract

The growth properties of a new panel of Bacillus subtilis tetL deletion strains and of a derivative set of strains in which tetL is restored to the chromosome support earlier indications that deletion of tetL results in a range of phenotypes that are unrelated to tetracycline resistance. These phenotypes were not reversed by restoration of a tetL gene to its native locus and were hypothesized to result from secondary mutations that arise when multifunctional tetL is deleted. Such genetic changes would temper the alkali sensitivity and Na(+) sensitivity that accompany loss of the monovalent cation/proton activity of TetL. Microarray comparisons of the transcriptomes of wild-type B. subtilis, a tetL deletion strain, and its tetL-restored derivative showed that 37 up-regulated genes and 13 down-regulated genes in the deletion strain did not change back to wild-type expression patterns after tetL was returned to the chromosome. Up-regulation of the citM gene, which encodes a divalent metal ion-coupled citrate transporter, was shown to account for the Co(2+)-sensitive phenotype of tetL mutants. The changes in expression of citM and genes encoding other ion-coupled solute transporters appear to be adaptive to loss of TetL functions in alkali and Na(+) tolerance, because they reduce Na(+)-coupled solute uptake and enhance solute uptake that is coupled to H(+) entry.

FIG. 1.

Schematic diagram of the tetL locus. The middle line depicts the tetL locus, indicating the tetL leader peptide CDS (hatched box) and tetL CDS (open box). The promoter (P) and terminator (t) are indicated. Relevant restriction sites are shown. The top line represents replacement of the tetL locus with the cat cassette in JC112 and JC112C. The bottom line represents replacement of the tetL leader and tetL CDS with a cat CDS in strains JC203, JC205, and JC206. The location and direction of transcription of genes upstream and downstream of tetL are indicated, as well as the site of insertion of an Sp resistance gene in the upstream yyaM gene.

FIG. 2.

Northern blot analysis of selected transcripts, as indicated on the left, from the strains indicated above the lanes. The fold change in each strain, relative to BD99, is shown below each lane. Data are averages from two experiments.

FIG. 3.

Alkalinization of the medium during growth. BD99, JC112, and JC112-R were grown in Spiz-KM medium (pH 8.0) for 6 h, to early stationary phase. Bars show the increase in pH determined in five independent experiments, with the error bars showing standard deviations. An analysis of variance, followed by pairwise comparison using the Bonferroni method, showed that the differences between BD99 and JC112 and between BD99 and JC112R were statistically significant (P < 0.001).

FIG. 4.

Carbon source-dependent growth patterns and effects of deleting citM on growth yield and Co²⁺ sensitivity. A. BD99, JC112, and JC112-R were grown in Spiz-KM (pH 7.0) to which only the indicated carbohydrate carbon sources were added. Growth yield, measured by A₆₀₀, was recorded after 16 h. B. BD99 (black bars), BD99 ΔcitM (open bars), JC112 (hatched bars), and JC112 ΔcitM (stippled bars) were grown for 8 h on Spiz-KM with no additions or containing 200 μM added CoCl₂. All experiments were conducted at least twice in duplicate. Error bars indicate standard deviations.

FIG. 5.

Ion-coupled membrane transporters that are hypothesized to change adaptively upon tetL deletion from B. subtilis. A. Schematic representation of the transport activities of JC112 that are deleted (tetL), down-regulated (nrgA and maeN), and up-regulated (citM, rocC, and rocE). Also shown is the change in czcD and nhaC, whose up-regulation was below the threshold of the current study but was shown earlier by transcriptional fusion and Northern blot analyses (51). B. Schematic representation of the transport activities of the two alternate l-malate transporters (34), MleN (53) and CimH (31, 32), that are hypothesized to take over for reduced maeN in support of the remaining capacity of JC112 for growth on malate. See text for discussion.