N. elongata Produces Type IV Pili That Mediate Interspecies Gene Transfer with N. gonorrhoeae

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species

Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior

Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. Delta pilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by PilT(L201C)). Purified PilT(L201C) forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae PilT(L201C) cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of PilT(L201C) cells is intermediate between the behaviors of wt and Delta pilT cells. The infection behavior of PilT(L201C) is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se, is not sufficient for N. gonorrhoeae micro-colony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology. IMPORTANCE Type IV pili are fibers expressed on the surface of many bacteria. Neisseria gonorrhoeae cells crawl, take up DNA, and communicate with each other and with human cells by retracting these fibers. Here, we show that an N. gonorrhoeae mutant expressing an enzymatically weakened type IV pilus retraction motor still crawls and takes up DNA normally. However, mutant cells exhibit abnormal social behavior, and they are less infective because they fail to activate the epidermal growth factor receptor. Our study shows that N. gonorrhoeae social and infection behaviors are sensitive to the strength of the retraction motor enzyme.HHS|NIH| National Institute of Allergy and Infectious Diseases (NIAID) [R01AI107966]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A bacterial filter protects and structures the gut microbiome of an insect

Associations with symbionts within the gut lumen of hosts are particularly prone to disruption due to the constant influx of ingested food and non-symbiotic microbes, yet we know little about how partner fidelity is maintained. Here we describe for the first time the existence of a gut morphological filter capable of protecting an animal gut microbiome from disruption. The proventriculus, a valve located between the crop and midgut of insects, functions as a micro-pore filter in the Sonoran Desert turtle ant (Cephalotes rohweri), blocking the entry of bacteria and particles ⩾0.2 μm into the midgut and hindgut while allowing passage of dissolved nutrients. Initial establishment of symbiotic gut bacteria occurs within the first few hours after pupation via oral–rectal trophallaxis, before the proventricular filter develops. Cephalotes ants are remarkable for having maintained a consistent core gut microbiome over evolutionary time and this partner fidelity is likely enabled by the proventricular filtering mechanism. In addition, the structure and function of the cephalotine proventriculus offers a new perspective on organismal resistance to pathogenic microbes, structuring of gut microbial communities, and development and maintenance of host–microbe fidelity both during the animal life cycle and over evolutionary time.This work was funded by NIH grant 5K12GM000708-13. PAPR received support from NSF Grant 0604067 to DEW.Open access.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Un état de la recherche sur la noblesse castillane à la fin du Moyen Age : Beceiro Pita (Isabel) y Cordoba de la Llave (Ricardo), Parentesco, poder y mentalidad. La nobleza castellana, siglos XII-XV, Madrid, C.S.I.C., 1990

Leroy Béatrice. Un état de la recherche sur la noblesse castillane à la fin du Moyen Age : Beceiro Pita (Isabel) y Cordoba de la Llave (Ricardo), Parentesco, poder y mentalidad. La nobleza castellana, siglos XII-XV, Madrid, C.S.I.C., 1990. In: Annales du Midi : revue archéologique, historique et philologique de la France méridionale, Tome 103, N°195, 1991. pp. 379-380

Transfer of antibiotic resistance marker between <i>N. elongata</i> and <i>N. gonorrhoeae</i>.

a<p>Number of rif^R recipient bacteria/total number of recipient bacteria (see Methods for differential selection of each species.).</p><p>*No growth of <i>N. gonorrhoeae</i> on LB Lennox agar.</p><p>**No growth of <i>N. elongata</i> on GCB/VCN agar. Values represent the average from three independent experiments ± SEM.</p

<i>N. elongata</i> produces Type IV pili.

<p>Scanning Electron Microscopy (SEM) of Wt <i>N. elongata</i>, (A), (B) and (C) and <i>N. elongata</i>Δ<i>pilE</i> (D). (C) is an enlarged image of the upper left hand section in (B). Arrowheads indicate Tfp-like fibers. Scale bars: 2 µm. (E) SDS PAGE of fibers isolated from wt <i>N. elongata</i> and <i>N. elongata</i>Δ<i>pilE</i> using a protocol for isolating <i>N. gonorrhoeae</i> Tfp. Arrow indicates the 17 kDa protein. (F) Top panel: Amino acid sequence deduced from the <i>N. elongata pilE</i> gene. Bottom panel: Sequences of peptides from the <i>N. elongata</i> 17 kD protein determined by tryptic digestion and MALDI-TOF mass spectroscopy. Deduced amino acid sequences that match the peptide sequences are in red.</p

Role of DUS in DNA transformation by wt <i>N. elongata.</i>

a<p>Transformation frequency is expressed as the number of rifampicin resistant bacteria/total CFUs. Values represent the average from three independent experiments ± SEM. “<” indicates the transformation frequency was below the limit of detection of the assay. “none”: medium only.</p

Transformation of <i>N. elongata</i> with rif^R chromosomal DNA.

a<p>Transformation frequency is expressed as the number of rif^R bacteria/total CFUs. Values are averaged from three independent experiments ± SEM. “<” indicates the transformation frequency was below the limit of detection of the assay.</p

Tfp biogenesis genes in <i>N. elongata</i> are transcribed.

<p>Migration of PCR amplicons generated from <i>N. elongata</i> cDNA using primers specific for <i>pilE</i>, <i>pilD</i>, <i>pilF</i>, and <i>pilQ</i>. (+) and (−) indicate the presence or absence of reverse transcriptase (RT) in the cDNA reaction.</p