The phosphate (P i ) starvation stimulon of Corynebacterium glutamicum was characterized by global gene expression analysis by using DNA microarrays. Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from P i -sufficient to P i -limiting conditions led to identification of five groups comprising 92 genes. Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium. One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4-to >30-fold-greater expression after the shift to P i limitation. Among these genes, the RNA levels of the pstSCAB (ABC-type P i uptake system), glpQ (glycerophosphoryldiester phosphodiesterase), ugpAEBC (ABC-type sn-glycerol 3-phosphate uptake system), phoH (unknown function), nucH (extracellular nuclease), and Cgl0328 (5-nucleotidase or related esterase) genes were increased, and pstSCAB exhibited a faster response than the other genes. Transcriptional fusion analyses revealed that elevated expression of pstSCAB and ugpAEBC was primarily due to transcriptional regulation. Several genes also involved in P uptake and metabolism were not affected by P i starvation; these included the genes encoding a PitA-like P i uptake system and a putative Na ؉ -dependent P i transporter and the genes involved in the metabolism of pyrophosphate and polyphosphate. In summary, a global, time-resolved picture of the response of C. glutamicum to P i starvation was obtained.Phosphorus (P) is an indispensable component of all cells in living organisms. In bacteria, P typically is assimilated as inorganic orthophosphate (P i ), which is transported into the cell by specific uptake systems. Alternatively, organophosphates and phosphonates may serve as sole P sources; either these compounds are imported by specific uptake systems and degraded intracellularly or P i that is liberated by extracellular degradation of the compounds is taken up into the cell. For several bacteria, particularly Escherichia coli and Bacillus subtilis, P metabolism and the regulatory mechanisms that permit adaptation to varying P availability have been well studied (22,47).E. coli possesses three P i uptake systems (19). PitA is expressed constitutively and transports phosphate in a proton motive force-dependent manner (47). When the extracellular P i concentration falls below about 4 M, P i is taken up primarily by the Pst system at the expense of ATP. The Pst system is an ABC transport system encoded by the pstSCAB genes of the pstSCAB-phoU operon, which is induced under P i starvation conditions (47). When formed, the PitB transporter encoded by a cryptic homolog of pitA is able to transport P i in a manner similar to the manner used by PitA (19). The genes constituting the P i starvation stimulon were identified in screening analyses based on transcriptional lacZ fusions (32) an...

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In the long-chain n-alkane degrader Acinetobacter sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two n-alkane hydroxylase-encoding genes in response to the chain length of n-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively.Several strains in the genus Acinetobacter are known as nalkane utilizers (4, 10). Among them, our isolate, Acinetobacter sp. strain M-1, is characterized by its ability to degrade a variety of n-alkanes, including very long chain n-alkanes (or paraffin wax) with carbon chain lengths of C 20 to C 44 that are in a solid state at ambient temperature (18).Several pathways have been proposed for the initial reaction of n-alkane degradation by Acinetobacter strains (1, 2, 4, 5). Previously, we demonstrated three n-alkane dioxygenase activities in Acinetobacter sp. strain M-1, which had been postulated by Finnerty (5). We assume that these enzymes are involved in the oxidation of n-alkanes that are slightly dissolved in the cytosol or oil inclusion of the cell, because the enzymes were found in the soluble fraction of the cell extract of strain M-1. Recently, the genes encoding alkane hydroxylase (alkM) (15), rubredoxin (rubA), and rubredoxin reductase (rubB) (8) in Acinetobacter calcoaceticus strain ADP1 were found, and each of the genes was shown to be indispensable for n-alkane degradation. These results suggest that a three-component alkane hydroxylase complex participates in n-alkane degradation in strain ADP1, which is similar to that in a medium-chain (C 6 to C 12 ) n-alkane degrader, Pseudomonas oleovorans (24). The difference in the organization of the genes involved in n-alkane degradation between P. oleovorans and A. calcoaceticus strain ADP1 is that these genes are dispersed over the chromosomal DNA in strain ADP1, while they form an operon on a large OCT plasmid in P. oleovorans.We describe here the isolation and characterization of genes in strain M-1 that are homologous to alkM, rubA, and rubB of strain ADP1. The most characteristic feature of strain M-1 was that two genes encoded alkane hydroxylases and they were differentially induced in response to the chain length of nalkanes.Cloning of two alkane hydroxylase genes, alkMa and alkMb, from Acinetobacter sp. strain M-1. We intended to clone the alkane hydroxylase-encoding gene from Acinetobacter sp. strain M-1 to study the molecular basis of the alkane hydroxylase complex in this organism. We designed the PCR primers mono-N and mono-C (Table 1) based on the highly conserved regions between alkM of A. calcoaceticus strain ADP1 (15) and alkB of P. oleovorans (11) and used the chromosomal DNA of strain M-1 as a template. This PCR yielded a 790-bp DNA fragment, and the sequence of the fragment was identical to a part of the alkMa gene (see below). Southern blot analysis using the fragment as the probe revealed that the probe hybridized to at least two bands in the genomic DNA of strain M-1 that had been digested with various restri...

Corynebacterium glutamicum contains genes for 13 two-component signal transduction systems. In order to test for their essentiality and involvement in the adaptive response to phosphate (P i ) starvation, a set of 12 deletion mutants was constructed. One of the mutants was specifically impaired in its ability to grow under P i limitation, and therefore the genes lacking in this strain were named phoS (encoding the sensor kinase) and phoR (encoding the response regulator). DNA microarray analyses with the C. glutamicum wild type and the ⌬phoRS mutant supported a role for the PhoRS system in the adaptation to P i starvation. In contrast to the wild type, the ⌬phoRS mutant did not induce the known P i starvation-inducible ( psi) genes within 1 hour after a shift from P i excess to P i limitation, except for the pstSCAB operon, which was still partially induced. This indicates an activator function for PhoR and the existence of at least one additional regulator of the pst operon. Primer extension analysis of selected psi genes ( pstS, ugpA, phoR, ushA, and nucH ) confirmed the microarray data and provided evidence for positive autoregulation of the phoRS genes.Phosphorus (P) is an essential nutrient for all cells and required, e.g., for the biosynthesis of nucleotides, DNA, and RNA and in addition for the functional regulation of protein activity by phosphorylation. The common phosphorus source is inorganic phosphate (P i ), and cells have developed mechanisms for the acquisition, assimilation, and storage of phosphate. Under phosphate starvation, many bacteria induce the synthesis of proteins that enable them to use the limiting phosphate resources more efficiently and to make alternative phosphorus sources accessible. The corresponding genes are collectively named P i starvation-inducible genes, or psi genes. The phosphate starvation response, in particular its regulation, has been most carefully studied in Escherichia coli (33) and Bacillus subtilis (11). In both species, two-component signal transduction systems consisting of a histidine kinase and a response regulator play a prominent role.In E. coli, induction of the P i starvation genes is dependent on the PhoR-PhoB two-component system. Under P i limitation, the histidine kinase PhoR phosphorylates the response regulator PhoB, and PhoBϳP in turn activates transcription of at least 31 genes, which form the Pho regulon (33). The genes include the phoBR operon; the pstSCAB-phoU operon, encoding an ABC transporter for high-affinity P i uptake and a regulatory protein; the ugpBAECQ operon, encoding an sn-glycerol 3-phosphate ABC uptake system and glycerophosphoryl diester phosphodiesterase; the phoA-psiF operon, encoding alkaline phosphatase and a protein of unknown function; phoE, encoding an anion-specific porin; phoH, encoding an ATPbinding protein of unknown function; and the phnCDEFGHI JKLMNOP operon, encoding proteins involved in the uptake of phosphonates and their degradation via the C-P lyase pathway. Thus, when P i is scarce, E. coli takes up P i by an ...

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h^–1 and biomass yields up to 0.47 g dry weight (g ethanol)^–1. Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.