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Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginineinducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.Arginine metabolism is of considerable significance in Pseudomonas aeruginosa, which can utilize this amino acid as a good source of carbon, nitrogen, and energy (16). The significance of arginine as a nutrient to P. aeruginosa is reflected in its being one of the strongest chemotactic attractants for this organism (10). This significance is also reflected in the presence of four catabolic pathways for the utilization of arginine ( Fig. 1) (16): the arginine deiminase pathway, the arginine succinyltransferase (AST) pathway; the arginine dehydrogenase pathway, and the arginine decarboxylase pathway.Recent work by Haas and coworkers has elucidated the role of anr (for anaerobic regulation) in induction of the arginine deiminase pathway by low oxygen tension (15). The arginine deiminase pathway functions to provide P. aeruginosa with energy in the absence of appropriate terminal electron acceptors (43). The anr gene of P. aeruginosa appears to be a close relative of fnr of Escherichia coli (49); in fact, the two genes can replace each other in heterologous systems (15). In contrast to the current understanding of the molecular basis for control of the arginine deiminase pathway, analogous information is lacking regarding the AST pathway (Fig. 1). This pathway, which converts arginine to glutamate, is considered the major route for catabolism in P. aeruginosa u...

The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic ␣-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.Bacillus subtilis secretes large amounts of proteins into the growth medium (43). Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by the Sec-dependent pathway, through which secretory proteins are synthesized as precursors with typical cleavable N-terminal signal peptides (3, 36). Fewer proteins are released into the medium via the cleavable twin-arginine translocation (TAT) system (39). Still other proteins are exported into the medium via ATP-binding cassette transporters, a dedicated pseudopilin export pathway, a competence development system or an ESAT-6 (Mycobacterium tuberculosis early secreted antigenic target of 6 kDa)-like system (31).The genome of B. subtilis 168 is 4,215 kbp in length and contains about 4,100 genes that are predicted to include over 250 extracellular proteins; the majority of these proteins are secreted through the aforementioned pathways (3, 18). However, proteomic studies have revealed that genome-based predictions reflect only 50% of the actual composition of the extracellular proteome. This significant discrepancy is mainly due to the difficulties in the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins (3, 18). These findin...

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Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major role in the control of certain biosynthetic and catabolic arginine genes, was purified to homogeneity. ArgR was shown to be a dimer of two equal subunits, each with a molecular mass of 37,000 Da. Determination of the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene. DNase I footprinting showed that ArgR protects a region of 45 to 47 bp that overlaps the promoters for the biosynthetic car and argF operons, indicating that ArgR exerts its negative control on the expression of these operons by steric hindrance. Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyltransferase pathway. Quantitative S1 nuclease experiments showed that expression of the first gene in this operon, aruC, is initiated from an arginine-inducible promoter. Studies with an aruC::lacZ fusion showed that this promoter is under the control of ArgR. DNase I experiments indicated that ArgR protects two 45-bp binding sites upstream of aruC; the 3 terminus for the downstream binding site overlaps the ؊35 region for the identified promoter. Gel retardation experiments yielded apparent dissociation constants of 2.5 ؋ 10 ؊11 , 4.2 ؋ 10 ؊12 , and 7.2 ؋ 10 ؊11 M for carA, argF, and aruC operators, respectively. Premethylation interference and depurination experiments with the car and argF operators identified a common sequence, 5-TGTCGC-3, which may be important for ArgR binding. Alignment of ArgR binding sites reveals that the ArgR binding site consists of two half-sites, in a direct repeat arrangement, with the consensus sequence TGTCGCN 8 AAN 5 .We reported, in the adjoining paper, the cloning and characterization of a gene, argR, that plays a major role in control of biosynthesis and aerobic catabolism of arginine in Pseudomonas aeruginosa (19). The argR gene is the sixth and terminal gene of an operon for the transport of arginine and ornithine (12,19,20). The derived amino acid sequence for ArgR exhibits significant homology to the AraC/XylS family of regulatory proteins (19). Inactivation of argR by gene replacement resulted in abolition of the arginine repression of the biosynthetic operons, car and argF, encoding carbamoylphosphate synthetase (CPS) and anabolic ornithine carbamoyltransferase (aOTC), respectively (19). The argR inactivation also resulted in abolition of the arginine induction of enzymes of the arginine succinyltransferase pathway (19), which is considered the major route for catabolism of arginine by P. aeruginosa under aerobic conditions (9). Consistent with this role for ArgR, the inactivated derivative was unable to utilize arginine or ornithine as the sole carbon source (19). This paper describes the purification and characterization of the regulatory protein encoded by argR. Interactions of the homogeneous protein with the control regions for the car, argF, and aru operons were investigated by various footprinting approaches. MATE...