Expression, purification and oligomerization of the S-adenosylmethionine transporter.

The S-adenosylmethionine carrier (SAMC) is a membrane transport protein located on the inner membrane of mitochondria that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix. SAMC mutations can cause a series of mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation and biosynthesis. Here, we describe the expression, purification and oligomerization of SAMC. The SAMC genes from three species were cloned into a eukaryotic expression vector with a GFP tag, and confocal microscopy analysis showed that these SAMCs were localized to mitochondria. A BacMam expression system was used for the expression of D. rerio SAMC with a FLAG tag. A size-exclusion chromatography analysis showed that SAMC may form a hexamer. A negative-staining electron microscopy analysis showed that SAMC formed tiny uniform particles and also confirmed the oligomerization of SAMC.

Broad-Spectrum Amino Acid Transporters ClAAP3 and ClAAP6 Expressed in Watermelon Fruits.

Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) through the phloem. The amino acid transporters involved in the import of Cit, Arg, and their precursors into developing fruits of watermelon have not been reported. In this study, we have compiled the list of putative amino acid transporters in watermelon and characterized transporters that are expressed in the early stage of fruit development. Using the yeast complementation study, we characterized ClAAP3 (Cla023187) and ClAAP6 (Cla023090) as functional amino acid transporters belonging to the family of amino acid permease (AAP) genes. The yeast growth and uptake assays of radiolabeled amino acid suggested that ClAAP3 and ClAAP6 can transport a broad spectrum of amino acids. Expression of translational fusion proteins with a GFP reporter in Nicotiana benthamiana leaves confirmed the ER- and plasma membrane-specific localization, suggesting the role of ClAAP proteins in the cellular import of amino acids. Based on the gene expression profiles and functional characterization, ClAAP3 and ClAAP6 are expected to play a major role in regulation of amino acid import into developing watermelon fruits.

Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB.

The global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY null mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY null mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Engineering photosynthetic production of L-lysine.

L-lysine and other amino acids are commonly produced through fermentation using strains of heterotrophic bacteria such as Corynebacterium glutamicum. Given the large amount of sugar this process consumes, direct photosynthetic production is intriguing alternative. In this study, we report the development of a cyanobacterium, Synechococcus sp. strain PCC 7002, capable of producing L-lysine with CO2 as the sole carbon-source. We found that heterologous expression of a lysine transporter was required to excrete lysine and avoid intracellular accumulation that correlated with poor fitness. Simultaneous expression of a feedback inhibition resistant aspartate kinase and lysine transporter were sufficient for high productivities, but this was also met with a decreased chlorophyll content and reduced growth rates. Increasing the reductant supply by using NH4+, a more reduced nitrogen source relative to NO3-, resulted in a two-fold increase in productivity directing 18% of fixed carbon to lysine. Given this advantage, we demonstrated lysine production from media formulated with a municipal wastewater treatment sidestream as a nutrient source for increased economic and environmental sustainability. Based on our results, we project that Synechococcus sp. strain PCC 7002 could produce lysine at areal productivities approaching that of sugar cane to lysine via fermentation using non-agricultural lands and low-cost feedstocks.

Cytokinins and Expression of SWEET, SUT, CWINV and AAP Genes Increase as Pea Seeds Germinate.

Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT), amino acid permease (AAP), Sugar Will Eventually be Exported Transporter (SWEET), cell wall invertase (CWINV), cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L. We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration.

Effect of high-concentrate diet on amino acid transporter expression and milk quality in Holstein dairy cows.

In order to evaluate the effect of high-concentrate diet supplementation on milk protein content, six Holstein dairy cows were assigned into high-concentrate diet (HC) or low-concentrate diet (LC) groups (N = 3/group) for 50 days. With regard to milk protein, HC feeding significantly reduced the percentage of milk protein (P < 0.01), and milk protein yield also reduced. The milk somatic cell count numbers and N-acetyl-D-glucosaminidase activity was significantly higher (P < 0.01) in the HC group than in the LC group. A pre-column derivatization procedure of o-phthalaldehyde was used to analyze the milk amino acid profile, the contents of Asp, Gln, Ala, Ile, Leu, and Lys were significantly lower in milk (P < 0.05), but Arg and Phe were significantly higher (P < 0.05) in the HC group than in the LC group. The mRNA abundance for amino acid transporters SLC7A8, SLC7A10 (P < 0.05), SLC1A3 (P < 0.05), and SLC16A10 (P < 0.05) were decreased in the HC group. These data indicate that expression of amino acid transporters alters regulation of amino acid utilization and decreases milk quality in dairy cows.

Disturbed intestinal nitrogen homeostasis in a mouse model of high-fat diet-induced obesity and glucose intolerance.

The oligopeptide transporter peptide cotransporter-1 Slc15a1 (PEPT1) plays a major role in the regulation of nitrogen supply, since it is responsible for 70% of the dietary nitrogen absorption. Previous studies demonstrated that PEPT1 expression and function in jejunum are reduced in diabetes and obesity, suggesting a nitrogen malabsorption from the diet. Surprisingly, we reported here a decrease in gut nitrogen excretion in high-fat diet (HFD)-fed mice and further investigated the mechanisms that could explain this apparent contradiction. Upon HFD, mice exhibited an increased concentration of free amino acids (AAs) in the portal vein (60%) along with a selective increase in the expression of two AA transporters (Slc6a20a, Slc36a1), pointing to a specific and adaptive absorption of some AAs. A delayed transit time (+40%) and an increased intestinal permeability (+80%) also contribute to the increase in nitrogen absorption. Besides, HFD mice exhibited a 2.2-fold decrease in fecal DNA resulting from a reduction in nitrogen catabolism from cell desquamation and/or in the intestinal microbiota. Indeed, major quantitative (2.5-fold reduction) and qualitative alterations of intestinal microbiota were observed in feces of HFD mice. Collectively, our results strongly suggest that both increased AA transporters, intestinal permeability and transit time, and changes in gut microbiota are involved in the increased circulating AA levels. Modifications in nitrogen homeostasis provide a new insight in HFD-induced obesity and glucose intolerance; however, whether these modifications are beneficial or detrimental for the HFD-associated metabolic complications remains an open issue.

Amino acid transporters expression in acinar cells is changed during acute pancreatitis.

Pancreatic acinar cells accumulate amino acids against a marked concentration gradient to synthesize digestive enzymes. Thus, the function of acinar cells depends on amino acid uptake mediated by active transport. Despite the importance of this process, pancreatic amino acid transporter expression and cellular localization is still unclear. We screened mouse pancreas for the expression of genes encoding amino acid transporters. We showed that the most highly expressed transporters, namely sodium dependent SNAT3 (Slc38a3) and SNAT5 (Slc38a5) and sodium independent neutral amino acids transporters LAT1 (Slc7a5) and LAT2 (Slc7a8), are expressed in the basolateral membrane of acinar cells. SNAT3 and SNAT5, LAT1 and LAT2 are expressed in acinar cells. Additional evidence that these transporters are expressed in mature acinar cells was gained using acinar cell culture and acute pancreatitis models. In the acute phase of pancreatic injury, when acinar cell loss occurs, and in an acinar cell culture model, which mimics changes occurring during pancreatitis, SNAT3 and SNAT5 are strongly down-regulated. LAT1 and LAT2 were down-regulated only in the in vitro model. At protein level, SNAT3 and SNAT5 expression was also reduced during pancreatitis. Expression of other amino acid transporters was also modified in both models of pancreatitis. The subset of transporters with differential expression patterns during acute pancreatitis might be involved in the injury/regeneration phases. Further expression, localization and functional studies will follow to better understand changes occurring during acute pancreatitis. These findings provide insight into pancreatic amino acid transport in healthy pancreas and during acute pancreatitis injury.

Transcriptional cross-regulation between Gram-negative and gram-positive bacteria, demonstrated using ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum.

The protein-gene pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in E. coli the activation of lysE by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP(d)) variants that confer elevated Arg-independent argO expression, some (ArgP(d)-P274S, -S94L, and, to a lesser extent, -P108S) activated lysE expression in E. coli. However, the individual activating effects of LysG and ArgP(d) on lysE were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP(d) variants exhibited higher affinity of binding to the lysE regulatory region and less DNA bending at both argO and lysE. We conclude that the transcription factor LysG from a Gram-positive bacterium, C. glutamicum, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium, E. coli, for activation of its cognate target lysE in vivo and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target lysE, but by a subtly different mechanism that renders them noninterchangeable with LysG.

Regulation of arginine acquisition and virulence gene expression in the human pathogen Streptococcus pneumoniae by transcription regulators ArgR1 and AhrC.

In this study, we investigated for the first time the transcriptional response of the human pathogen Streptococcus pneumoniae to fluctuating concentrations of arginine, an essential amino acid for this bacterium. By means of DNA microarray analyses, several operons and genes were found, the expression of which was affected by the concentration of arginine in the medium. Five of the identified operons were demonstrated to be directly repressed in the presence of high arginine concentrations via the concerted action of the ArgR-type regulators ArgR1 and AhrC. These ArgR1/AhrC targets encompass the putative amino acid transport genes artPQ, abpA, abpB, and aapA; the arginine biosynthetic genes argGH; and the virulence genes aliB and lmB/adcAII-phtD encoding an oligopeptide-binding lipoprotein and cell surface Zn(2+)-scavenging units, respectively. In addition, the data indicate that three of the amino acid transport genes encode an arginine ATP-binding cassette transporter unit required for efficient growth during arginine limitation. Instead of regulating arginine biosynthetic and catabolic genes as has been reported for other Gram-positive bacteria, our findings suggest that the physiological function of ArgR1/AhrC in S. pneumoniae is to ensure optimal uptake of arginine from the surrounding milieu.

New insights into the signaling mechanism of the pH-responsive, membrane-integrated transcriptional activator CadC of Escherichia coli.

The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.

Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene.

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

Dietary amino acid supplementation affects temporal expression of amino acid transporters and metabolic genes in selected and commercial strains of rainbow trout (Oncorhynchus mykiss).

Replacement of fishmeal as the major protein source in feeds is critical for continued growth and sustainability of the aquaculture industry. However, numerous studies have shown suboptimal fish growth performance and reduced protein retention efficiency when carnivorous fish species are fed low fishmeal-high plant protein feeds. A study was conducted using a commercial strain and a genetically improved strain of rainbow trout selected for improved performance when fed an all plant protein diet to identify physiological differences associated with growth performance in the selected trout strain. Fifty individuals per strain (average weight ~ 580 g) were force-fed a plant-protein blend with and without amino acid supplementation (lysine, methionine and threonine) at 0.5% body weight and sampled at intervals over 24 h. Samples from intestine and liver were analyzed for specific gene expression analysis related to amino acid transporters, digestive process control, protein degradation and amino acid metabolism. The results showed that expression levels of various intestinal amino acid transporters (SLC1A1, SLC7A9, SLC15A, SLC1A5 SLC6A19 and SLC36A1) were affected by strain, diet and time. Moreover, significant interactions were found regarding the temporal expression levels of cholecystokinin (CCK-L), Krüppel-like factor 15 (KLF15) and aspartate aminotransferase (GOT) transcripts in the examined tissues. The results provide evidence that improved growth and protein retention of the selected strain fed an all-plant protein diet is a result of nutritional adaptation and an overall change in physiological homeostatic control.

Transport of the photodynamic therapy agent 5-aminolevulinic acid by distinct H+-coupled nutrient carriers coexpressed in the small intestine.

5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.

Downregulation of cystine transporter xc by irinotecan in human head and neck cancer FaDu xenografts.

BACKGROUND: The purpose of this study was: (1) to document the critical requirement of cystine for growth of human tumor cells in vitro, and (2) to determine the effect of the anticancer agent irinotecan on the cystine transporter x(c)(-) in head and neck FaDu xenografts. METHODS: Cell growth was measured by sulforhodamine B assay. xCT protein, glutathione (GSH) and DNA damage were determined using Western blot, spectrophotometry, and immunohistochemistry, respectively. RESULTS: Depletion of cystine from the medium inhibited tumor cell growth. Treatment of FaDu tumor with a therapeutic dose of irinotecan resulted in depression of xCT protein levels, leading to tumor growth retardation and downregulation of GSH with increased reactive oxygen species (ROS). The accumulation of ROS correlated with increased DNA damage as evidenced by increased H2AX. CONCLUSION: Depression of xCT protein by irinotecan resulted in downregulation of GSH and increase in ROS, which could be the other possible mechanisms of DNA damage by irinotecan.

Regulation of amino acid transporters in the rat remnant kidney.

BACKGROUND: Partial renal ablation is associated with compensatory renal growth, significant azotaemia, a significant increase in fractional excretion of sodium and changes in solute transport. The present study evaluated the occurrence of adaptations in the remnant kidney, especially in renal amino acid transporters and sodium transporters and their putative role in sodium handling in the early stages (24 h and 1 week) after uninephrectomy. METHODS: Wistar rats aged 8 weeks old were submitted to renal ablation of the right kidney--Unx rats (n = 10). 24 hours (n = 5) and 1 week (n = 5) after surgery, rats were anesthetized and the left kidney was removed. Urinary and plasmatic levels of catecholamines, sodium, urea and creatinine were measured. Gene expression of the amino acid and sodium transporters was determined by Real-time reverse transcription PCR. Protein expression was evaluated by Western blot using specific antibodies for the amino acid and sodium transporters. RESULTS: Uninephrectomized (Unx) rats for 24 h showed a lower urinary excretion of L-DOPA, dopamine and DOPAC than the corresponding Sham rats, accompanied by an increase in the expression of the Na(+)-K(+)-ATPase protein (64% increase). Unx rats for 1 week presented a hypertrophied remnant kidney, higher urine outflow and a approximately 2-fold increase in the fractional excretion of sodium. The NHE3 mRNA expression was significantly decreased in Unx rats throughout the study (approximately 20% decrease). LAT1 transcript and protein were consistently overexpressed at both 24 h and 1 week after uninephrectomy. In contrast, 4F2hc and LAT2 transcript abundance was lower in 24-h Unx rats than in Sham rats (a 36% decrease in both cases). CONCLUSIONS: These results provide evidence that the renal expression of the amino acid transporters LAT1, LAT2 and 4F2hc and the sodium transporters Na(+)-K(+)-ATPase and NHE3 is differently regulated following unilateral nephrectomy. In conclusion, this study allowed us to characterize the renal adaptations in the early stages after uninephrectomy, which showed a combined interaction of multiple mechanisms regulating sodium homeostasis including the renal dopaminergic system, and the abundance of amino acid transporters and sodium transporters.

Induction of cadBA in an Escherichia coli lysine auxotroph transformed with a cad-gfp transcriptional fusion.

CadBA functions as a part of overall Escherichia coli response to low extracellular pH. A gfpmut3 structural gene transcriptionally fused to the cadBA promoter (Pcad) was used as a reporter to monitor changes in intracellular lysine as a potential factor influencing cadBA induction. Different patterns of cadBA induction were observed in two E. coli strains with different lysine biosynthetic capabilities. In E. coli ZK126 (pJBA25-Pcad), a lysine prototroph, maximum levels of induction were detected 3 h after the transfer of bacterial cells under inducing conditions (pH 5.8; 3.4 microM extracellular lysine). The induction subsequently decreased until hour 7 after which no further change in expression was observed. However, in the lysine depleted strain E. coli ATCC 23812 (pJBA25-Pcad) which is an auxotroph for lysine, no decrease in cadBA expression was observed over time under the same induction conditions. Although no time dependent statistical differences in intracellular lysine were observed, bacterial cells depleted for no longer than 4 h (1.38 +/- 0.25 micromol lysine/g cell dry weight) exhibited more rapid induction of cadBA (after 3 h) and a lower maximum level of induction compared to cells with relatively lower intracellular lysine (approximately 1.08 micromol/g cell dry weight). For the latter, the detectable level of induction was delayed for 1 h but the maximum level of induction response was higher.

HU participates in expression of a specific set of genes required for growth and survival at acidic pH in Escherichia coli.

The major histone-like Escherichia coli protein, HU, is composed of alpha and beta subunits respectively encoded by hupA and hupB in Escherichia coli. A mutant deficient in both hupA and hupB grew at a slightly slower rate than the wild type at pH 7.5. Growth of the mutant diminished with a decrease in pH, and no growth was observed at pH 4.6. Mutants of either hupA or hupB grew at all pH levels tested. The arginine-dependent survival at pH 2.5 was diminished approximately 60-fold by the deletion of both hupA and hupB, whereas the survival was slightly affected by the deletion of either hupA or hupB. The mRNA levels of adiA and adiC, which respectively encode arginine decarboxylase and arginine/agmatine antiporter, were low in the mutant deficient in both hupA and hupB. The deletion of both hupA and hupB had little effect on survival at pH 2.5 in the presence of glutamate or lysine, and expression of the genes for glutamate and lysine decarboxylases was not impaired by the deletion of the HU genes. These results suggest that HU regulates expression of the specific set of genes required for growth and survival in acidic environments.

Regulation of an amino acid transporter mRNA in Trypanosoma brucei.

Trypanosoma brucei regulates gene expression by post-transcriptional mechanisms, such as mRNA turnover and translation control. This regulation frequently requires specific sequences located in the 3'-untranslated region. Microarray analysis and Northern blot hybridization showed that the amino acid transporter 11 mRNA is up-regulated in insect stages of the parasite. By RT-PCR and sequencing, the AATP11 polyadenylation site was mapped. We show that this 3'-UTR causes higher expression of the chloramphenicol acetyltransferase (CAT) reporter gene in procyclic trypanosomes than in bloodstream forms. Results of deletion analysis suggested that multiple elements located between nucleotides 141 and 618 of the 3'-untranslated region are required for this control.