A revised model for the control of fatty acid synthesis by master regulator Spo0A in Bacillus subtilis.

In starving Bacillus subtilis cells, the accDA operon encoding two subunits of the essential acetyl-CoA carboxylase (ACC) has been proposed to be tightly regulated by direct binding of the master regulator Spo0A to a cis element (0A box) in the promoter region. When the 0A box is mutated, biofilm formation and sporulation have been reported to be impaired. Here, we present evidence that two 0A boxes, one previously known (0A-1) and another newly discovered (0A-2) in the accDA promoter region are positively and negatively regulated by Spo0A∼P respectively. Cells with mutated 0A boxes experience slight delays in sporulation, but eventually sporulate with high efficiency. In contrast, cells harboring a single mutated 0A-2 box are deficient for biofilm formation, while cells harboring either a mutated 0A-1 box or both mutated 0A boxes form biofilms. We further show that the essential ACC enzyme localizes on or near the cell membrane by directly observing a functional GFP fusion to one of the enzyme's subunits. Collectively, we propose a revised model in which accDA is primarily transcribed by a major σA -RNA polymerase, while Spo0A∼P plays an additional role in the fine-tuning of accDA expression upon starvation to support proper biofilm formation and sporulation.

Mutational analyses reveal overall topology and functional regions of NilB, a bacterial outer membrane protein required for host association in a model of animal-microbe mutualism.

The gammaproteobacterium Xenorhabdus nematophila is a mutualistic symbiont that colonizes the intestine of the nematode Steinernema carpocapsae. nilB (nematode intestine localization) is essential for X. nematophila colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by nilA and nilC, it confers upon noncognate Xenorhabdus spp. the ability to colonize S. carpocapsae nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient-rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, 14 transmembrane strands, and seven extracellular surface loops and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB and, by extension, its homologs in mucosal pathogens.

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes.

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

A bifunctional DNA binding region in Tn5 transposase.

Tn5 transposition is a complicated process that requires the formation of a highly ordered protein-DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein-DNA contacts, although many protein-DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase-DNA binding events.

Evidence for "unseen" transposase--DNA contacts.

In this study, evidence of novel, important interactions between a hyperactive Tn5 transposon recognition end sequence and hyperactive Tn5 transposase (Tnp) are presented. A hyperactive Tn5 end sequence, the mosaic end (ME), was isolated previously. The ME and a wild-type end sequence, the outside end (OE), differ at only three positions, yet transposition on the ME is tenfold higher than on the OE in vivo. Also, transposition on the ME is much more efficient than transposition on the OE in vitro. Here, we show that the decreased activity observed for the OE is caused by a defect in paired ends complex (PEC) formation resulting from the orientation of the A-T base-pair at position 4 of this end. Efficient PEC formation requires an interaction between the C5-methyl group (C5-Me) on the non-transferred strand thymine base at position 4 (T4) and Tnp. PEC formation on nicked substrates is much less affected by the orientation of the A-T base-pair at position 4, indicating that the C5-Me group is important only for steps preceding nicking. A recently determined co-crystal structure of Tn5 Tnp with the ME is discussed and a model explaining possible roles for the base-pair at position 4 is explored.