Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen. Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and ␥-aminobutyrate (GABA). We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism. We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC. A ⌬gabDT mutant grew normally on all of the nitrogen sources tested except GABA. The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes. Nac was required for gab transcription in vivo and in vitro. Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally. A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor. GabC is proposed to reduce nitrogen source-specific modulation of expression. Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required S . Previous studies showing S -dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains. The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction. We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis.

Previously, using a chromosomal reversion assay system, we established that an adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under stress, and we demonstrated that multiple mechanisms are involved in generating these mutations (41, 43). In an attempt to delineate how these mutations are generated, we began an investigation into whether or not transcription and transcription-associated proteins influence adaptive mutagenesis. In B. subtilis, the Mfd protein (transcription repair coupling factor) facilitates removal of RNA polymerase stalled at transcriptional blockages and recruitment of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An association between Mfd mutagenesis and aspects of transcription is discussed.Since the mid-1950s, microbiologists have been aware of mutations occurring in nondividing populations of cells (22,29). The formation of these mutants was alternatively termed "starvation-associated mutagenesis" (29), "adaptive mutation" (8), or "stationary-phase mutagenesis" (13). Recently, variations of this phenomenon have been investigated with Escherichia coli (8,29,38), Pseudomonas (33), Bacillus subtilis (41), and the eukaryotic yeast Saccharomyces cerevisiae (15,40). These phenomena reveal that starving populations of cells can acquire mutations favoring growth after the application of selection.While the phenomenon of stationary-phase mutation is widespread, it is clear that the mechanism(s) by which it arises varies from organism to organism. To date, the most favored system for studying adaptive or stationary-phase mutagenesis is the RecA-dependent E. coli FC40 system investigated by, among others, the laboratories of Cairns, Foster, Rosenberg, and Roth (7,8,11,18). Recent results strongly suggest that this mutagenesis is the result of gene amplification followed by mutation in a transiently growing population of cells (18). In light of this, we chose to investigate the possibility that transiently growing cells may play a role in a B. subtilis system containing three chromosomal point mutations. The phenomenon of transcriptional mutagenesis, or retromutagenesis, whereby RNA polymerase bypasses an unrepaired DNA lesion or otherwise produces an altered mRNA, which is then translated into a protein of altered function, could provide a transient growth advantage for the cell. This mechanism has been proposed for other model systems, including eukaryotes (6, 9).We have previously shown the existence of one such mutagenic phenomenon occurring during stationary phase in B. subtilis cells starved for amino acids (41). This mutagenic process appears to enhance the survivability of cell populations undergoing nutritional stress. In brief, isogenic strains of B. subtilis carrying three amino acid auxotrophies conferred by hisC952 (amber), metB5 (ochre), and leuC427 (missense) are incubated on medium lacking one of the required amino acids. After several days of incubati...

Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427, under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu ؉ reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.The generation of mutations has been traditionally ascribed to spontaneous processes affecting actively growing, dividing cells. Nevertheless, by the mid-1950s, several reports describing mutagenesis in nondividing cells of bacteria, plants, flies, and fungi appeared in the scientific literature (reference 36 and references therein). Much of the initial characterization of this process in bacteria took place in the laboratory of Francis Ryan, who observed Escherichia coli mutants capable of synthesizing histidine arising from his mutant (auxotrophic) cells undergoing prolonged starvation (36) while cell turnover remained undetectable, and DNA replication slowed with increasing time (26). Renewed interest in adaptive mutation was generated when Cairns and coworkers published their work on the generation of Lac ϩ reversions in E. coli cells unable to use the lactose provided as the sole carbon source in a minimal medium (6). This work demonstrated that adaptive mutations can arise as a result of stress rather than from selection of preexisting mutations. The generation of stress-induced Lac ϩ reversions, assayed via a plasmid-borne system, has been studied intensively by several laboratories (reviewed in references 13, 15, and 34; 32) and is dependent on activation of the SOS and/or stress responses. Further studies have also suggested that a subpopulation within the Lac Ϫ stressed cells engage in an exquisitely regulated transient state of hypermutation limited in time and to DNA sites near double-stranded DNA breaks (reviewed in reference 15). Collectively, the results from studies on this system have provided interesting insights into the acquisition of beneficial mutations and demonstrated the role of several genetic factors in the adaptive mutation phenomenon.

The discovery of antimicrobial peptides (AMPs) has brought tremendous promise and opportunities to overcome the prevalence of bacterial resistance to commonly used antibiotics. However, their widespread use and translation into clinical application is hampered by the moderate to severe hemolytic activity and cytotoxicity. Here, we presented and validated a supramolecular platform for the construction of hemo- and cytocompatible AMP-based nanomaterials, termed self-assembling antimicrobial nanofibers (SAANs). SAANs, the "nucleus" of our antimicrobial therapeutic platform, are supramolecular assemblies of de novo designed AMPs that undergo programmed self-assembly into nanostructured fibers to "punch holes" in the bacterial membrane, thus killing the bacterial pathogen. In this study, we performed solid-state NMR spectroscopy showing predominant antiparallel β-sheet assemblies rather than monomers to interact with liposomes. We investigated the mode of antimicrobial action of SAANs using transmission electron microscopy and provided compelling microscopic evidence that self-assembled nanofibers were physically in contact with bacterial cells causing local membrane deformation and rupture. While effectively killing bacteria, SAANs, owing to their nanoparticulate nature, were found to cross mammalian cell membranes harmlessly with greatly reduced membrane accumulation and possess exceptional cytocompatibility and hemocompatibility compared to natural AMPs. Through these systematic investigations, we expect to establish this new paradigm for the customized design of SAANs that will provide exquisite, tunable control of both bactericidal activity and cytocompatibility and can potentially overcome the drawbacks of traditional AMPs.

Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames ( M oraxella catarrhalis is a Gram-negative mucosal pathogen that has attracted increased interest within the scientific and medical communities for its role in several clinically significant human infections. The bacterium is a cause of upper respiratory tract infections including sinusitis and otitis media in healthy children (10, 17, 62). More recently, M. catarrhalis has been shown to be involved in conjunctivitis in children (9) and in acute exacerbations of chronic sinusitis in adults (11). Additionally, in adults, it is an important etiologic agent of exacerbations of chronic obstructive pulmonary disease (COPD) (54,55,62). It has been estimated that M. catarrhalis is responsible for up to 10% of exacerbations of COPD in the United States, a finding which translates into as many as 4 million infections per year (43).For M. catarrhalis to cause clinical disease, it typically must spread from its initial site of colonization in the nasopharynx into either the middle ear or the lower respiratory tract. It is believed that biofilm formation is an important event involved in colonization of the nasopharynx, and a recent study demonstrated that M. catarrhalis was present in a biofilm in the middle ear of children with chronic otitis media (25). It is likely that M. catarrhalis exists in a biofilm together with other normal flora in the nasopharynx. Until relatively recently, no studies had been performed in an in vivo environment to identify and better characterize the bacterial factors involved with colonization of the nasopharynx by M. catarrhalis. However, utilizing a chinchilla model, Luke et al. (36) demonstrated that type IV pili are important for colonization by M. catarrhalis in this animal model.Previous studies have examined the human antibody response to known surface proteins of M. catarrhalis as a surrogate for identification of bacterial genes expressed in vivo (for a representative example, see reference 42), and one study was able to detect mRNA from a small number of selected M. catarrhalis genes in nasopharyngeal secretions from young children with acute respiratory tract illness (39). The demonstration that the chinchilla nasopharynx can be colonized by M. catarrhalis (5, 36), together with the development of M. catarrhalis DNA microarrays (19,65), presented the op...