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FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia , and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1 , plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter- lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia . In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2 . The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae , and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC . In P. unamae , the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

Open comedones from thirty-eight patients with acne vulgaris on the face or back were compared for microbial flora. A total of eighty-three comedones from the face and sixty-three from the upper back were individually processed for quantitative bacterial analysis. The greatest difference between the flora of comedones at these two sites was that 44.6% of comedones from the back (compared to 9.6% from the face) harboured no aerobic cocci. The decreased prevalence of staphylococci in the lesions from the back reflects the relative absence of these organisms in isolated follicles from normal uninvolved skin of the back. The geometric mean count of anaerobes in comedones from the back was in the same range as the count found in isolated follicles in normal uninvolved skin in a previous study. This work supports the concept that the bacterial flora of comedones is an extension of the follicular flora and may be unrelated to the event of comedogenesis.

We have investigated the mechanisms underlying prostaglandin inhibition of histamine-stimulated parietal cell function. Enzyme-dispersed canine parietal cells were enriched by elutriation. The accumulation of the weak base [14C]aminopyrine was used as an index of parietal cell function and cyclic adenosine monophosphate content was measured by radioimmunoassay. Step density gradients of the elutriator-enriched parietal cell fractions indicated that parietal cells accounted for the histamine stimulation of cyclic adenosine monophosphate production and inhibition by the prostaglandin E analogue Enprostil. Pertussis toxin adenosine diphosphate-ribosylates a subunit with a molecular weight of 41,000, thereby inactivating the inhibitory guanine nucleotide-binding protein of adenylate cyclase. Pertussis toxin treatment of parietal cells in overnight suspension culture was used to determine if inhibitory guanosine triphosphate-binding protein mediated prostanoid inhibition. In control cultured cells, prostaglandin E2 and Enprostil markedly inhibited forskolin- and histamine-stimulated aminopyrine accumulation. In parietal cells treated with pertussis toxin (300 ng/ml) for 18 h, stimulation of parietal cell function by histamine, isobutylmethylxanthine, and forskolin was unaltered compared with control cells, whereas prostaglandin E2 and Enprostil inhibition was markedly reduced. In pertussis toxin-treated cells, histamine-stimulated cyclic adenosine monophosphate generation was unaltered, whereas Enprostil inhibition of histamine-stimulated cyclic adenosine monophosphate production was markedly reduced. Pertussis toxin treatment of membranes from control, but not from pertussis toxin-treated, cells induced the [32P]adenosine diphosphate-ribosylation of a membrane protein with a molecular weight of 41,000, presumably the alpha-subunit of inhibitory guanosine triphosphate-binding protein. We conclude that prostanoids inhibit parietal cell function by receptor-mediated interaction with the inhibitory guanine nucleotide-binding protein of adenylate cyclase.

Patients with severe nodulo-cystic acne are known to have elevated serum antibody levels and increased immediate hypersensitivity reactions to Propionibacterium acnes. This organism is the predominant bacterium in normal pilosebaceous follicles of human skin, and can be consistently isolated from pustular lesions in acne. Previously it had been observed that delayed cutaneous hypersensitivity reactions to P. acnes were negative in patients with acne. The present study investigated the proliferative response of lymphocytes from patients with nodulo-cystic acne to phytohaemagglutinin (PHA) and P. acnes antigen stimulation. The response to PHA stimulation was within normal limits. The response to P. acnes antigen showed a significant increase over control values obtained by testing lymphocytes from acne-free subjects. Thus cell mediated immunity to P. acnes may be present in subjects with severe inflammatory acne. These findings raise the possibility that reactions to P. acnes may contribute to intensifying the inflammatory response in acne lesions.

A technique for quantitating bacteria in isolated pilosebaceous follicles is described. This involves microdissection of the follicles from biopsies of skin, using the method of chemical pretreatment of skin to facilitate the separation of the epidermis and epidermal appendages from the dermis. The aerobic cocci and anaerobic diphtheroids in pilosebaceous follicles in 66 biopsies of scalp and 48 biopsies of skin of the upper back were quantitated using this technique. On the back, aerobic staphylococci were very sparse in normal follicles, indicating that their primary habitat on the skin must be on the skin surface rather than within follicles. Of 138 isolated follicles from skin of the upper back, 94 contained no aerobic cocci. Anaerobic organisms were present in high numbers within normal follicles. The geometric mean density of anaerobes in 138 isolated follicles from skin of the upper back was 3.8 X 10(4) diphtheroids per follicle. Eighty-eight follicles contained more than 10(4) anaerobic diphtheroids. Using data from scalp biopsies we found that there was a correlation between the weight of sebaceous glands and the density of anaerobes within the follicles attached to these glands (coefficient of correlation = 0.6).

Cellular mechanisms underlying the anti-secretory actions of the prostaglandin E2 analogue enprostil were studied using enzyme-dispersed, elutriator-enriched canine parietal cells and the accumulation of the weak base 14C-labeled aminopyrine as a functional index. Enprostil inhibited the accumulation of aminopyrine stimulated by histamine and the phosphodiesterase inhibitor isobutylmethyl, but not by carbachol, gastrin, or dibutyryl cyclic adenosine monophosphate. Inhibition by enprostil was dose-dependent (0.1 nM to 1 microM), with maximal inhibition ranging from 65 to 95 percent. Over the same concentration range, enprostil inhibited the histamine-stimulated generation of cyclic adenosine monophosphate. This selective inhibition of histamine activation of parietal cell function was comparable to that found for prostaglandin E2. Forskolin, a diterpene that directly activates the catalytic subunit of adenylate cyclase, was also markedly inhibited by nanomolar concentrations of prostaglandin E2 and enprostil. We conclude that at least a component of the secretory inhibition by enprostil reflects direct interference with histamine stimulation of parietal cell adenylate cyclase.