The pathogenic Neisseria species are human-adapted pathogens that cause quite distinct diseases. Neisseria gonorrhoeae causes the common sexually transmitted infection gonorrhea, while Neisseria meningitidis causes a potentially lethal form of bacterial meningitis. During infection, both pathogens deploy a number of virulence factors in order to thrive in the host. The focus of this review is on the outer membrane transport systems that enable the Neisseriae to utilize host-specific nutrients, including metal-binding proteins such as transferrin and calprotectin. Because acquisition of these critical metals is essential for growth and survival, understanding the structures of receptor-ligand complexes may be an important step in developing preventative or therapeutic strategies focused on thwarting these pathogens. Much can also be learned by comparing structures with antigenic diversity among the transporter sequences, as conserved functional domains in these essential transporters could represent the pathogens' "Achilles heel." Toward this goal, we present known or modeled structures for the transport systems produced by the pathogenic Neisseria species, overlapped with sequence diversity derived by comparing hundreds of neisserial protein sequences. Given the concerning increase in N. gonorrhoeae incidence and antibiotic resistance, these outer membrane transport systems appear to be excellent targets for new therapies and preventative vaccines.

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of mdc genes that enable a soil bacterium, Acinetobacter baylyi ADP1, to use malonate as a carbon source. Despite previous assumptions that the mdc-gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of mdcR, a regulatory gene that is convergent to other mdc genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate mdc-gene transcription. Although mdcR deletion prevented growth on malonate, a 1 nt substitution in the promoter of mdcA enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5'-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in A. baylyi ADP1.

These data suggest that common temperate sponges can be a source of bioactive chemical and microbial diversity. Further studies may reveal the importance of the microbial associates to the sponge and natural product biosynthesis.

SummaryProteobacteria often co-ordinate responses to carbon sources using CRP and the second messenger cyclic 3′, 5′-AMP (cAMP), which combine to control transcription of genes during growth on non-glucose substrates as part of the catabolite-repression response. Here we show that cAMP-CRP is active and important in Vibrio fischeri during colonization of its host squid Euprymna scolopes. Moreover, consistent with a classical role in catabolite repression, a cAMP-CRPdependent reporter showed lower activity in cells grown in media amended with glucose rather than glycerol. Surprisingly though, intracellular cAMP levels were higher in glucose-grown cells. Mutant analyses were consistent with predictions that CyaA was responsible for cAMP generation, that the EIIA Glc component of glucose transport could enhance cAMP production and that the phophodiesterases CpdA and CpdP consumed intracellular and extracellular cAMP respectively. However, the observation of lower cAMP levels in glycerol-grown cells seemed best explained by changes in cAMP export, via an unknown mechanism. Our data also indicated that cAMP-CRP activity decreased during growth on glucose independently of crp's native transcriptional regulation or cAMP levels. We speculate that some unknown mechanism, perhaps carbon-source-dependent post-translational modulation of CRP, may help control cAMP-CRP activity in V. fischeri.

Neisseria gonorrhoeae and Neisseria meningitidis are human-specific pathogens in the Neisseriaceae family that can cause devastating diseases. Although both species inhabit mucosal surfaces, they cause dramatically different diseases. Despite this, they have evolved similar mechanisms to survive and thrive in a metal-restricted host. The human host restricts, or overloads, the bacterial metal nutrient supply within host cell niches to limit pathogenesis and disease progression. Thus, the pathogenic Neisseria require appropriate metal homeostasis mechanisms to acclimate to such a hostile and ever-changing host environment. This review discusses the mechanisms by which the host allocates and alters zinc, manganese, and copper levels and the ability of the pathogenic Neisseria to sense and respond to such alterations. This review will also discuss integrated metal homeostasis in N. gonorrhoeae and the significance of investigating metal interplay.

Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ. Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg 2ϩ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), D-alanine, or D-glutamate, respectively. We hypothesized that NAG, D-alanine, or D-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for ␦-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.KEYWORDS Photobacterium, Aliivibrio, symbiosis, aminolevulinic acid, hemin, photobacteria D efined knockout mutant libraries are useful resources that have been generated to promote research in several bacterial experimental models and pathogens (1-10). In such studies, researchers have often listed essential genes, the knockouts of which were apparently lethal under the conditions of library construction and therefore not represented by mutants in the library. In order to recover insertion mutants collectively

Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal "on" expression when PhoB is active, minimal background in the "off" state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at nineteen additional intervening nucleotide positions for which we did not predict sequence-specific effects the bases were randomized. Eighty-three such constructs were screened in , enabling us to identify bases at particular randomized positions that significantly correlated with high "on" or low "off" expression. A second round of promoter design rationally constrained thirteen additional positions, leading to a reporter with high PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized GFP, the latter with a half-life of eighty-one minutes in In culture, PhoB induced the reporter when phosphate was depleted below 10 μM. During symbiotic colonization of its host squid , the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and re-engineering. Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between "on" and "off" states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over thirty-four billion sequence variants, we identified bases correlated with favorable performance by screening only eighty-three candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of This promoter-design strategy will facilitate the engineering of other regulator-specific reporters.

TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae , the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo .