Concetta C. DiRusso

Multiple Factors Independently Regulate hilA and Invasion Gene Expression in Salmonella enterica Serovar Typhimurium

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting that hilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulate hilA expression. flhDC and fliA encode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhC and fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimuriums ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.

Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.

SUMMARY The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.

Mutational Analysis of a Fatty Acyl-Coenzyme A Synthetase Signature Motif Identifies Seven Amino Acid Residues That Modulate Fatty Acid Substrate Specificity

Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12-C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWXPXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431NGWLHTGDIAVMDEEGFLRIVDRKK455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.

The Structural Basis of Acyl Coenzyme A-Dependent Regulation of the Transcription Factor Fadr

FadR is an acyl‐CoA‐responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo‐protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl‐CoA. The recently described structure of apo‐FadR shows a DNA binding domain coupled to an acyl‐CoA binding domain with a novel fold, but does not explain how binding of the acyl‐CoA effector molecule >30 Å away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadR‐operator and FadR‐myristoyl‐CoA binary complexes. The FadR‐DNA complex reveals a novel winged helix‐turn‐helix protein‐DNA interaction, involving sequence‐specific contacts from the wing to the minor groove. Binding of acyl‐CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 Å. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 Å in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl‐CoA‐responsive regulation.

Crystal structure of FadR, a fatty acid‐responsive transcription factor with a novel acyl coenzyme A‐binding fold

FadR is a dimeric acyl coenzyme A (acyl CoA)‐binding protein and transcription factor that regulates the expression of genes encoding fatty acid biosynthetic and degrading enzymes in Escherichia coli. Here, the 2.0 Å crystal structure of full‐length FadR is described, determined using multi‐wavelength anomalous dispersion. The structure reveals a dimer and a two‐domain fold, with DNA‐binding and acyl‐CoA‐binding sites located in an N‐terminal and C‐terminal domain, respectively. The N‐terminal domain contains a winged helix–turn–helix prokaryotic DNA‐binding fold. Comparison with known structures and analysis of mutagenesis data delineated the site of interaction with DNA. The C‐terminal domain has a novel fold, consisting of a seven‐helical bundle with a crossover topology. Careful analysis of the structure, together with mutational and biophysical data, revealed a putative hydrophobic acyl‐CoA‐binding site, buried in the core of the seven‐helical bundle. This structure aids in understanding FadR function at a molecular level, provides the first structural scaffold for the large GntR family of transcription factors, which are keys in the control of metabolism in bacterial pathogens, and could thus be a possible target for novel chemotherapeutic agents.

The fats of Escherichia coli during infancy and old age: regulation by global regulators, alarmones and lipid intermediates

The fluidity and phase state of bacterial lipid bilayers commonly change in response to ambient environmental conditions to maintain the critical functions of the envelope as a semipermeable and selective boundary. A special, and intricate, set of alterations in membrane lipid metabolism is elicited by conditions causing growth arrest. Under such conditions, specific alterations in the membrane lipid–fatty acid composition are required for survival of the cell and, concurrently, the membrane lipids are suggested to serve as endogenous reserves providing carbon/energy for maintenance requirements. It appears that the global regulator FadR is required for both of these activities to be performed properly and that the FadR regulon is interconnected to the universal stress response of Escherichia coli. FadR, in conjuction with long‐chain fatty acyl‐CoA, long‐chain acyl‐ACP, ppGpp and cAMP, are key players in regulating the activities of enzymes and expression of genes involved in fatty acid and phospholipid metabolism in dividing and ageing E. coli cells.

Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism

Previously we demonstrated that supplementation with the polyunsaturated fatty acids (PUFA) arachidonic acid (AA) or docosahexaenoic acid (DHA) increased neurite outgrowth of PC12 cells during differentiation, and that overexpression of rat acyl-CoA synthetase long-chain family member 6 (Acsl6, formerly ACS2) further increased PUFA-enhanced neurite outgrowth. However, whether Acsl6 overexpression enhanced the amount of PUFA accumulated in the cells or altered the partitioning of any fatty acids into phospholipids (PLs) or triacylglycerides (TAGs) was unknown. Here we show that Acsl6 overexpression specifically promotes DHA internalization, activation to DHA-CoA, and accumulation in differentiating PC12 cells. In contrast, oleic acid (OA) and AA internalization and activation to OA-CoA and AA-CoA were increased only marginally by Acsl6 overexpression. Additionally, the level of total cellular PLs was increased in Acsl6 overexpressing cells when the medium was supplemented with AA and DHA, but not with OA. Acsl6 overexpression increased the incorporation of [14C]-labeled OA, AA, or DHA into PLs and TAGs. These results do not support a role for Acsl6 in the specific targeting of fatty acids into PLs or TAGs. Rather, our data support the hypothesis that Acsl6 functions primarily in DHA metabolism, and that its overexpression increases DHA and AA internalization primarily during the first 24 h of neuronal differentiation to stimulate PL synthesis and enhance neurite outgrowth.

Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids: Amino acid residues within the ATP/AMP signature motif of Escherichia coli fadD are required for enzyme activity and fatty acid transport

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. InEscherichia coli this enzyme, encoded by thefadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif (213YTGGTTGVAKGA224and 356GYGLTE361) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25–45% wild type activity (fadD G216A, fadD T217A,fadD G219A, andfadD K222A) and 2) those with 10% or less wild-type activity (fadD Y213A,fadD T214A, andfadD E361A). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.

Two different pathways are involved in the β-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications

In Pseudomonas putida U, the degradation of n‐alkanoic and n‐phenylalkanoic acids is carried out by two sets of β‐oxidation enzymes (βI and βII). Whereas the first one (called βI) is constitutive and catalyses the degradation of n‐alkanoic and n‐phenylalkanoic acids very efficiently, the other one (βII), which is only expressed when some of the genes encoding βI enzymes are mutated, catabolizes n‐phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the βI genes handicaps the growth of P. putida U in media containing n‐alkanoic or n‐phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n‐alkanoates as a result of the induction of βII, but they were still unable to catabolize n‐phenylalkanoates completely, as the βI‐FadBA enzymes are essential for the β‐oxidation of certain n‐phenylalkanoyl‐CoA derivatives when they reach a critical size. Owing to the existence of the βII system, mutants lacking βIfadB/A are able to synthesize new poly 3‐OH‐n‐alkanoates (PHAs) and poly 3‐OH‐n‐phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.

Bacterial Long Chain Fatty Acid Transport: Gateway to a Fatty Acid-responsive Signaling System

Exogenous long chain fatty acids (LCFAs) influence a myriad of cellular processes including intracellular signaling and patterns of gene expression. Some cell types accumulate distinct classes of LCFAs whereas others accumulate or release fatty acids following some type of external stimulus (i.e. hormonal or nutritional cues). Given the energetic cost of fatty acid synthesis on one hand and (at least in the mammalian system) the necessity for obtaining essential fatty acids from the environment, the transport of LCFAs must necessarily represent a fundamental biological process. The biochemical mechanisms underpinning fatty acid transport involve specific proteins and enzymes, which act either directly at the membrane or indirectly at the level of downstream metabolism. Distinct membrane-bound and membrane-associated fatty acid transport proteins have been identified and characterized in a number of different systems including bacteria, yeast, and mammals (1–7). The genetic and biochemical foundations of LCFA transport were first elucidated in the Gram-negative bacterium Escherichia coli. The hallmark of this system is the coupled transport and activation of exogenous LCFAs, which in turn leads to changes in transcription patterns of the genes encoding the proteins required for fatty acid biosynthesis and degradation. The three central components of this system include FadL, an outer membrane-bound fatty acid transport protein, FadD, an inner membrane-associated long chain acyl-CoA synthetase (ACSL), and FadR, a long chain acyl-CoA-responsive transcription factor. The bacterial fatty acid transport and trafficking system leading to downstream fatty acid-responsive transcriptional regulation serves as a fundamental paradigm in biology, which provides a number of guiding principles that can be applied to more complex systems.