The glutamic acid decarboxylase and putative GABA:glutamate antiporter system plays a prominent role in E. coli acid resistance. While it might seem that regulation of this system should be straightforward with acid pH inducing expression and alkaline pH preventing induction, regulation of the gadA and gadBC operons is much more complex. Several reports, some seemingly contradictory, have invoked various roles for CRP and H-NS as repressors, σS and σ70 as principal sigma factors, and an AraC-like regulator, GadX, as both inducer and repressor. The data presented in this study concern another AraC-like regulatory protein, GadW, and its collaborative role with GadX in controlling this system prior to and during entry into stationary phase. The study also examines CRP-mediated control of this system. The results add more clarity to the complex model of gadA and gadBC expression and its regulation. Figure summarizes the basic control circuits mapped for this system.
FIG. 10. Model of the GadX and -W and CRP-RpoS control circuit. GadX activates expression from PgadA, while GadW inhibits expression (in the presence of GadX) from PgadA and PgadX. Transcription of the activator gene gadX is largely dependent on the RpoS sigma (more ...)
An earlier report describing GadX as a repressor at pH 8 but an activator at pH 5.5 can now be partly explained (18
). The activator-repressor model was based on data obtained from cells grown in minimal glucose media. The repressor effect can also be seen, although less dramatically, in the LB-grown cells represented in Fig. . More GadA and -B protein is seen at pH 8 in the gadX
mutant (Fig. , lane 3) than is found in the wild type (Fig. , lane 1), originally suggesting that GadX was a repressor at pH 8. The inducer effect at pH 5.5 can be seen in Fig. , where the gadX
mutant (Fig. , lane 4) produces less GadA and -B protein than does the wild type (Fig. , lane 2), suggesting that GadX was an activator at pH 5.5. However, when GadW is missing, GadX is really only a positive regulator (Fig. , lanes 5 and 6). Repression at pH 8 appears to be due to an effect of pH on GadW function (Fig. , lanes 3 and 4).
The fact that GadX and GadW can each independently activate gadA
under certain conditions indicates that both proteins are capable of binding to the gadA
promoter regions, a conclusion supported in vitro for both GadX (18
) and GadW (this report). The data also shows that GadW regulates the gadX
promoter (Fig. ). The effects of GadW on gadA
expression are not solely through its impact on gadX
expression, however, since GadW can affect gadA
expression even in the absence of GadX (Fig. , compare lanes 3 and 4 with lanes 7 and 8). An interesting question is why does GadW, in the absence of GadX, activate gadA
only at pH 8? One model, discussed briefly above, is that GadW might bind gadA
promoters best when cells are grown at pH 8, which either prevents GadX from binding or attenuates GadX activation. Alternatively, GadW may not strongly bind the gadA
promoter regions if GadX is already bound to them. In this model, GadW, under ordinary circumstances, might primarily temper GadX activity by repressing gadX
The transcriptional organization of the gadA
locus involves three promoters, one upstream of gadA
, one upstream of gadX
, and one upstream of gadW
. Transcription from PgadA
can produce a cotranscript with gadX
, but transcription from PgadX
does not yield a cotranscript with gadW
) (this report). However, mutations that affect expression from PgadA
have a large effect on gadA
message levels but almost no effect on the gadAX
cotranscript (Fig. and for gadW
effects and Fig. for crp
effects). This suggests either that gadA
are usually transcribed separately and only occasionally form cotranscripts or that the gadAX
cotranscript is subject to vigorous processing that separates the two messages.
Consistent with their annotation as AraC-like regulators, GadX and GadW will readily form homodimers in vivo (5
). The finding that these proteins also appear capable of forming heterodimers may provide insight into the GadX and -W control circuit. Heterodimers could be incorporated into a model that explains GadW inhibition of GadX activity, although equally plausible alternative hypotheses involving competitive DNA binding are possible. Many AraC-like regulators bind ligands that alter their function (5
), but whether or not GadX or GadW binds specific cytoplasmic ligands remains unknown.
Expression of gadA
in LB media is principally dependent on RpoS. A recent study, confirmed here, demonstrated that expression of gadX
is RpoS dependent (20
). In that same study, gadX
was uncoupled from RpoS control by placing gadX
under the control of a Tn5
promoter-operator element. When gadX
was expressed from this promoter, the gadA
genes were transcribed even in log-phase (pH 7.4), LB-grown cells, a condition ordinarily devoid of gadA
expression. This finding suggested that the RpoS effects on gadA
expression are indirect, occurring due to an RpoS requirement for gadX
expression. The result also suggests that the gadA
promoters are themselves RpoS independent. The fact that expression of gadA
in minimal glucose media is mostly RpoS independent and GadX independent supports that idea (data not shown). However, the result also suggests that another gadA
induction pathway exists, one independent of GadX.
We have also found that CRP represses gadX expression, which explains the derepressive effect that crp mutations have on gadA and gadBC expression. CRP appears to negatively regulate gadX expression by virtue of its effects on RpoS production. The developing model for the control circuit is that, when E. coli is actively growing under conditions where cAMP levels are high (e.g., exponential growth in media like LB medium), CRP will repress gadX by inhibiting RpoS production. GadW also represses gadX under this condition. As cells approach stationary phase, RpoS levels rise, causing increased expression of gadX. GadX levels increase and overcome repression of gadA and gadBC by GadW. Cells grown fermentatively on glucose would have lower cAMP levels and less CRP-dependent repression of the system.
The pH control of this system, at least in rich, undefined media, also appears tied to the pH control of RpoS production. Evidence supporting this model includes the fact that gadA
expression in this media requires RpoS and that the levels of glutamate decarboxylase and of RpoS are induced earlier in pH 5.5 cultures entering stationary phase than in pH 8 cultures. It has previously been shown that pH influences translation of rpoS
message and degradation of RpoS protein in Salmonella enterica
The nucleoid protein H-NS has also been identified as a repressor for this system (4
). A recent report has demonstrated that H-NS may work to repress the gadA
genes by repressing gadX
). However, our results, using a different genetic background, indicate that H-NS still affects gadA
expression in a gadXW
mutant. Thus, H-NS must also act on gadA
independently of gadX
. Another inconsistency between the two studies is that an hns
mutation in our K-12 strain did not relieve CRP-dependent repression of gadA
Thus, our results argue that CRP, not H-NS, is a master regulator of glutamate-dependent acid resistance. The reason(s) for these discrepancies is not apparent and may be strain dependent.
The question remains: why does the cell expend so much energy to regulate the gad genes? At present count there are two repressors (H-NS and CRP), one activator (GadX), one repressor-activator (GadW), and two sigma factors focused on controlling this system. This complex regulatory network must reflect the importance that E. coli places on surviving transient exposures to extreme acid stress, ensuring that the system is in place under any environmental condition that could lead to acid stress. Numerous questions remain regarding the regulatory interactions that take place between these many factors.