As an enzyme of the tricarboxylic acid cycle pathway, citrate synthase participates in the generation of a variety of cellular biosynthetic intermediates and in that of reduced purine nucleotides that are used in energy generation via electron transport-linked phosphorylation reactions. It catalyzes the condensation of oxaloacetate and acetyl coenzyme A to produce citrate plus coenzyme A. In Escherichia col this enzyme is encoded by the giA gene. To investigate how gitA expression is regulated, a gA4-lacZ operon fusion was constructed and analyzed following aerobic and anaerobic cell growth on various types of culture media. Under aerobic culture conditions, expression was elevated to a level twofold higher than that reached under anaerobic culture conditions. ArcA functions as a repressor of gtA expression under each set of conditions: in a 1arcA strain, glA-lacZ expression was elevated to levels two-and eightfold higher than those seen in a wild-type strain under aerobic and anaerobic conditions, respectively. This control is independent of the finr gene product, an alternative anaerobic gene regulator in E. coli. When the richness or type of carbon compound used for cell growth was varied, gltA-lacZ expression varied by 10-to 14-fold during aerobic and anaerobic growth. This regulation was independent of both the crp andfruR gene products, suggesting that another regulatory element in E. coli is responsible for the observed control. Finally, glA-lacZ expression was shown to be inversely proportional to the cell growth rate. These findings indicate that the regulation of giA gene expression is complex in meeting the differential needs of the cell for biosynthesis and energy generation under various cell culture conditions. The Escherichia coli gltA gene product, citrate synthase (EC 4.1.3.7), catalyzes the condensation of acetyl coenzyme A with oxaloacetate to form citrate. Because of its key position as the first enzyme of the tricarboxylic acid (TCA) cycle, citrate synthase had been assumed to be an important control point for determining the metabolic rate of the cell. On the basis of enzyme activities in wild-type and TCA cycle mutants, a branched pathway was proposed to operate under anaerobic conditions. In the absence of oxygen certain TCA cycle enzymes contribute significantly to cellular biosynthesis rather than to energy generation (18). Citrate synthase must function under both aerobic (TCA cycle) and anaerobic (branched pathway) conditions, as the cell does not possess an alternative enzyme at this step, in contrast to other steps of the cycle (e.g., fumarate reductase and succinate dehydrogenase, and the fumarases). Measurements of citrate synthase levels show that citrate synthase synthesis is suppressed by anaerobiosis and glucose and elevated by the presence of oxygen and acetate (10,30). Enzyme levels are inversely related to the growth rate (10,30).The E. coli enzyme is typical of citrate synthases of gramnegative microorganisms in that it is a large enzyme composed of six identical subunits ...

The degraded nucleic acids and ribosomes of its prey cell provide Bdelovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 ,uM in attack phase cells and 35 ,uM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated.

The intracellularly growing bacterium Bdellovibrio bacteriovorus 109J transports intact ATP by a specific, energy-requiring process. ATP transport does not involve either an ADP-ATP or ah AMP-ATP exchange mechanism but, instead, has characteristics of an active transport permease. Kinetically distinct systems for ATP transport are expressed by the two developmental stages of the bdellovibrio life cycle. In cell suspensions to which y-abeled ATP was added there was a roughly linear uptake Of 32p for 1 or 2 min that was not influenced by the addition of 0.1 mM potassium phosphate to the suspension (Fig. la). This indicated that the 32p label was not being transported after hydrolysis to Pi. In addition, the observed rate of [y32P]ATP accumulation (148 pmol/min per mg of protein) was almost identical to the rate of accumulation of ATP that was labeled with 3H in the adenine ring (164 pmol/min per mg of protein) (Fig. lb). These two results are consistent with the conclusion that bdellovibrio cells transport ATP as an intact molecule. The accumulation was a temperature-dependent process and was sensitive to respiratory inhibitors such as cyanide (Fig. la), azide, and the proton gradient uncoupler carbonyl cyanide m-chlorophenyl hydrazone (data not shown).To demonstrate that a compoutd is accumulated by active transport, it must be shown both that a radioactive label is concentrated intracellularly and that the label is in a chemically unaltered form. Over 94% of the total radioactivity taken up by bdellovibrios exposed to.[3H]ATP for 1 min was present in the trichloroacetic acid-soluble fraction of the cell contents. Chromatography of this fraction by reverse-phase high-performance liquid chromatography (19) revealed that essentially all of the label conmigrated with authentic ATP (Fig. 2). By using an internal volume of 1 4ul/1010 cells (19), it is possible to calculate that the internal concentration of labeled ATP after 1 min of accumulation was 5.5 times the concentration of ATP in the suspending medium. Thus bdellovibrios have the capacity to internally concentrate intact ATP against a gradient.During the course of their unique developmental life cycle bdellovibrios exist in two differentiated forms: an intracellular growth-phase cell and an extracellular attack-phase cell

Membrane-derived oligosaccharides (MDO), a class of osmotically active carbohydrates, are the major organic solutes present in the periplasm of Escherichia coli and many other gram-negative bacteria when cells are grown in a medium of low osmolarity. Analyses of growing cells of Bdellovibnro bacteriovorus, a gram-negative predator of other bacteria, have confirmned that they also synthesize a characteristic MDO-like class of oligosaccharides. The natural growth environment of bdellovibrios is the periplasm of other gram-negative bacteria. Because of this location, prey cell MDO constitute a potential source of organic nutrients for growing bdellovibrios. Using cells of E. coli whose MDO were 3H labeled, we examined the extent to which B. bacteriovorus 109J metabolizes these prey cell components. Interestingly, there was neither significant degradation nor incorporation of prey cell MDO by bdellovibrios during the course of their intracellular growth. In fact, bdellovibrios had little capability either to degrade extracellular MDO that was made available to them or to transport glucose, the major monomeric constituent of prey cell MDO. Instead, periplasmic MDO were irreversibly lost to the extracellular environment during the period of bdellovibrio attack and penetration. Thus, although prey cell periplasmic proteins are retained, other important periplasmic components are released early in the bdellovibrio growth cycle. The loss of these MDO may aid in the destabilization of the prey cell plasma membrane, increasing the availability of cytoplasmic constituents to the periplasmic bdellovibrio.

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