Previous work demonstrated that UFA biosynthesis in P. aeruginosa is governed by two pathways, depending on the oxygen availability: 1) two aerobic fatty acid desaturase pathways consisting of DesA and DesBC, and 2) the anaerobic FabAB pathway. The fabAB-encoded proteins play an essential and dominant role in the UFA biosynthesis under both aerobic and anaerobic conditions.
While the existence of and the mechanisms of UFA synthesis via the anaerobic pathway were known at the onset of the studies presented here, little was known about the regulation of expression of the fabA and fabB genes. It was known that, unlike E. coli where fabA and fabB map to two distinct locations on the chromosome, these two genes form an operon in P. aeruginosa. It was also known that the expression of this operon was up-regulated in biofilm-grown cells and repressed by addition of fatty acids, especially oleic acid, to the growth medium. While this study did not reveal any specific regulatory protein(s) involved in regulation of fabAB operon expression or the exact molecular mechanisms governing regulation of expression of this operon, they revealed some previously unknown findings.
First, RT-PCR analyses indicated that the fabAB operon is transcribed from at least two promoters. It is co-transcribed with the upstream, seemingly unrelated PA1612-PA1611 operon, but an additional promoter located in the PA1611-fabA intergenic region which also contributes to fabAB expression.
Second, the DesT (FabR) repressor binds to a palindromic sequence in the PA1611-fabA intergenic region, but seems to play only a modest role in the fabAB operon expression.
Third, a 30 bp sequence present in the PA1611-fabA intergenic region is a regulatory element involving the positive regulation of fabA. The position of this sequence at an appropriate position upstream of a putative -10 promoter consensus sequence indicates that it may be an activator-binding site.
Fourth, while E. coli FadR belongs to the GntR family of transcriptional regulators, deletion of none of the 25 genes encoding the GntR family of regulators in P. aeruginosa affected fabA transcription. Therefore, the P. aeruginosa FadR-like activator, if it exists, must belong to a different family of regulators. Additionally, deletion of the immediate upstream gene PA1611 encoding a hybrid sensor kinase/response regulator protein did not adversely affect fabA transcription, at least not under the conditions employed in the present studies.
, numerous other cellular factors including RpoN, DesA and Anr seem to play at least minor roles in regulation of fabAB
transcription, possibly through modulation of intracellular fatty acid levels or other metabolites. RpoN is a global regulator which regulates expression of nitrogen assimilation gene and fermentation gene expression (reviewed in 
). However, there is no evidence that rpoN
regulates UFA biosynthesis in P. aeruginosa
. If it does, it may be due to global effects. According to previous study, PA0286
was shown to encode DesA, an aerobic fatty acid desaturase 
. This gene might be closely linked to the regulation of the fabAB
operon by affecting cellular UFA levels. Enoyl-CoA hydratase/isomerases are involved in fatty acid metabolism. These hydratase activities catalyze the hydratation of 2-trans
-enoyl-CoA into 3-hydroxyacyl-CoA and the isomerase activities shift the 3- double bond of the intermediates of UFA oxidation to the 2-trans
position. The anr
gene product, which senses low oxygen, supports anaerobic growth by activating numerous genes. P. aeruginosa
is able to survive in an anaerobic environment. Since P. aeruginosa
grows as a biofilm-type in the anaerobic CF lung mucus, anaerobic conditions may be an important factor to regulate metabolic pathways for robust growth and establishment of persistent infections. The anaerobic survival mode is supported by denitrification of nitrate or nitrite 
. In addition, the arginine deaminase (ADI) pathway plays a key role in catabolizing L-arginine to L-ornithine, with the formation of ATP from ADP, resulting in the growth of P. aeruginosa
under anaerobic condition in the absence of terminal electron acceptors such as molecular oxygen or nitrate (reviewed in 
). Since Anr globally regulates expression of many genes, P. aeruginosa fabAB
regulation via Anr may be indirect via other Anr-dependent factors. An alternate explanation might be that Anr activity may be inhibited by another factor(s) which may be highly expressed under aerobic growth, but Anr expression from a high-copy number plasmid may be able to saturate this “anti-Anr” factor and thus complement the deletion mutant. Since Δanr
mutants are unable to grow anaerobically, additional experiments should be performed with cells grown under microaerophilic conditions to further assess the potential role of Anr in fabAB
gene expression. In conclusion, use of gene fusion technology revealed Anr as an activator of fabA′-lacZ
expression in E. coli
, but it played only a minor and probably indirect role in P. aeruginosa
. While none of these findings does yet provide a clear picture of the molecular mechanisms governing transcription of the fabAB
operon, they indicate that regulation of P. aeruginosa fabAB
operon expression is very complex and most likely quite different from what has been described in E. coli
, DINAMelt analysis suggests that fabAB
expression may be regulated not via protein-binding, but via a yet-to-be discovered mechanism. One possibility lies in small regulatory RNA (srRNA)-mediated regulation of fabAB
expression. srRNA can regulate gene expression at the transcriptional and translational levels. Intracellular metabolites such as amino acids, sugars and nucleotides can bind to cis
-acting metabolite-sensing regulatory RNA elements and control gene expression, which are called riboswitches (reviewed in 
). Various types of riboswitches are present in bacteria. Generally, non-coding regulatory RNA elements are found in the 5′-untranslated region (5′-UTR) and can give rise to three-dimensional conformational alternate changes in response to changes intracellular metabolite signals. Recently, it was found that besides metabolites, metals such as intracellular Mg2+
can regulate the Mg2+
transporter MgtA of Salmonella enterica
serovar Typhimurium by the metal-sensing 5′-UTR of the mgtA
. Furthermore, changes in environmental conditions such as temperature can result in a conformational change of regulatory RNA, thus functioning as a “thermometer”. For example, virulence genes are highly expressed at 37°C by a PrfA transcriptional activator, which is thermally regulated by the 5′UTR of mRNAs of prfA
in Listeria monocytogenes
. In addition, the ROSE (repressor of heat-shock gene expression) element placed in the 5′UTR region of heat-shock genes in many Gram-negative bacteria senses temperature changes in order to control their gene expression 
. E. coli rpoS
mRNA translation is regulated by the DsrA small RNA which disrupts a sequestering helix of a ribosome binding site during temperature downshift 
may employ a regulatory RNA for controlling anaerobic UFA synthesis. One probable pathway is that a metabolite-sensing non-coding RNA may recognize intracellular or exogenous UFA and change its structure, resulting in fabAB
gene repression. This is consistent with the observation that OA supplementation repressed fabA
expression up to 50% (). Another possible explanation is that low growth temperature might be an important controlling factor. Since cells should maintain the membrane fluidity for normal function required for transport and movement, they adapt to changes in environmental conditions, especially temperature. Upon exposure to low temperatures, cell membranes become solid-state, but they preserve their fluidity by increasing UFA levels. According to previous study, fabA
expression is highly up-regulated in cells grown at 16°C compared to cells grown at at 37°C, suggesting positive regulation by low temperature 
. Therefore, the 5′-UTR of fabA
may sense low temperature and coordinate an adaptation process caused by a temperature downshift. However, having said all of this, searches of the fabAB
upstream sequences have yet to reveal possible srRNAs.
As mentioned above, the fabAB operon is transcribed from at least two promoters, P1 and P2, resulting in transcription of two mRNAs, mRNA I and mRNA II (). In the absence of exogenous UFAs and under anaerobic, as well as perhaps low temperature conditions, the fabAB operon is transcribed from both of these promoters and the respective transcripts terminate at a single transcriptional terminator which is located immediately downstream of the fabAB operon (). Transcription from P2 requires an activator protein which binds to the 30 bp region. Maximal transcription and translation ensures an adequate supply of FabA and FabB proteins for UFA synthesis. At the mRNA level, this situation is characterized by a riboregulatory element configuration on mRNA I containing an antitermination element.
A working model for P. aeruginosa fabAB regulation.
In the presence of exogenous UFAs and/or aerobic or high (37°C) temperature conditions, the proposed activator probably binds an UFA-CoA ligand and the activator-UFA-CoA complex dissociates from the 30 bp activation element resulting in loss or of fabAB transcription from P2 (). At the mRNA level, this situation is characterized by a riboregulatory element configuration that contains a terminator. The result is a low level transcription and translation of fabA and fabB, a desirable situation when oxygen levels and temperatures are high, exogenous UFAs are available and the overall cellular demand for UFAs is either low or can be satisfied by the activities of the aerobic desaturase pathways.
While many of our experimental results support this model, several questions do remain unanswered. For example, why were repeated genetic and biochemical attempts aimed at identification of the proposed activator protein unsuccessful? Why does purified DesT bind to the PA1611
intergenic region, but there is little to no effect on fabA
expression in lacZ
fusion or microarray experiments? A possible explanation for this may be that most of the experiments described in this study were performed with cells grown aerobically and at 37°C, possibly conditions during which synthesis and/or activity of an activator protein is repressed or DesT activity is masked. This notion is supported by the fact that fabA′-lacZ
expression was significantly higher in cells grown at 16°C when compared to cells grown at 37°C. Future experiments should therefore be performed with cells grown under anaerobic or microaerophilic conditions, as well as with cells grown at lower temperatures. Although DINAMelt analysis suggests the possibility that a riboregulatory element may be involved in regulation of fabAB
operon expression, there is currently no experimental evidence supporting this possibility. Future efforts should therefore also include experiments that would address the existence of a 5′ UTR RNA with a high degree of secondary structure. The experiments would consist of in-line probing 
and RNase H cleavage assays of RNA-DNA hybrids 
in the presence and absence of substrate to determine if the structure not only exists, but also undergoes a conformational change upon addition of the substrate or changes in an environmental cue. The nature of the substrate(s) or environmental cue(s), of course, remains speculative. Substrates could be metabolites such as a UFA-CoAs and environmental cue signals such as low temperature or low oxygen levels.
Studies on anaerobic UFA synthesis by the P. aeruginosa FabAB pathway and identification of its regulatory signals are significant because many infections caused by this bacterium, especially cystic fibrosis lung infections, are biofilm-type infections during which this bacterium's physiology is adapted to an anaerobic lifestyle. Such studies have therefore the potential for discovering new targets and drugs for treatment of biofilm-type infections.