Gram + and Gram - promoters - I am embarassed to even ask this (Dec/07/2005 )

Ok so I am totally ashamed to even have to ask this but here goes. I am making a promoter gene construct in the hopes to express my gene in e. coli and then certain gram + bacteria as well. Question is (and I am sure this is too basic) if I will need different promoters for the two. I am using the Amp resistance gene promoter from pUC18 to drive my gene of interest. I thought of using the Lac Operon to control gene expression but I am sure this will bring up more factors that may interfere. Anyone have experience with this?

-Captain_DNA-

One promoter should work with the other. I think the transcriptional machinery is the same for most prokaryotic promoter sequences...if not, they sure lied to me back in mol biol in college

-aimikins-

Thanks aimikins, I needed a second opinion on this before going ahead and spending a lot of time trying to get it to work if it was doomed to failure. Like I said, it is a very basic bio question and somehow the answer escaped my mind, sob. Thanks for the input!

-Captain_DNA-

There is no need to be ashamed of asking this question...

The answer is, unfortunately, there is no way to tell, unless it's been done before and it's in the literature. For example, I work with B. fragilis, a Gram negative anaerobe that shares the same niche as E. coli, but E. coli promoters don't work in B. fragilis, and B. fragilis promoters don't work in E. coli.

Now, this is for routine housekeeping promoters. There are, of course, any number of different promoters in both E. coli and B. fragilis (and any other bacteria, Gram positive or negative, for that matter) that differ from the constitutive housekeeping promoter and rely on alternate sigma factors for their operation. Whether there's any overlap in these type of promoters between any two bacteria is unknown until tested...

So, unfortunately, you'll have to either try it and pray, or find something in the literature where they tried and either succeeded or failed at expressing a gene under your promoter in your target organism(s).

-HomeBrew-

You may want to look at the Pspac promoter in gram-positive.

QUOTE

The state of the art in controlled expression in Bacillus subtilis is the spac expression system, which is based on the application of the lac repressor-operator control system from Escherichia coli. In B. subtilis, the system uses a constitutive penicillinase promoter to express the lac repressor. Isopropyl--D-thiogalactopyranoside (IPTG)-dependent expression of the target gene is from a hybrid promoter-operator consisting of the SPO1 bacteriophage promoter and lac operator sequence (35). Despite its broad use for conditional expression in B. subtilis, the spac system has been widely recognized for its capacity to allow significant expression in the absence of an inducer. Indeed, only relatively recently has there been a systematic study of expression from the spac promoter, indicating demonstrable uninduced expression of lacZ from the spac promoter of the pMUTIN vector system (33).

http://aem.asm.org/cgi/content/full/67/1/403

I work with Strep. mutans and good inducible promoter in gram-positive are rare and sometimes does not work well from one species to another even in the same group. For example, one promoter can work well in Strep. pneumo but when we use it Step. mutans we get poor results.

-iansmith-

Thanks all so far. Yeah I am looking more for constituative promoter. Right now I am using the onee that drives Ampicillin resistance in the pUC plasmid family. Inducible would be nice too but right now I have to check to see if this one works first.

-Captain_DNA-

Would a promoter from a transposon be a good alternative? The promoter from transposon that can insert in several species tend to be more universal than promoter found on species/group specific plasmid or those from genomic DNA.

-iansmith-

wow, I sure didn't know what I was talking about....

I know that pACYC plasmids can be maintained in both E coli and S aureus, so I made the assumption that a constitutive promoter from a pUC plasmid would probably work in most bacteria...

I'm sorry I bombed on you, Captain DNA. I'm glad others were there to set it straight.

-aimikins-

No one is perfect Aimikins, not even...me . Heh heh

-Captain_DNA-