The vegetative cells of the filamentous cyanobacterium Nostoc punctiforme can differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893 N. punctiforme genes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH 4 ؉ -grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N 2 -grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N 2 fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up-and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N 2 -fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N 2 grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.

Hormogonia are nongrowing filaments, motile by means of a gliding mechanism, that are produced by certain cyanobacteria. Their differentiation is induced by positive and negative factors for growth, such as deprivation of combined nitrogen (nitrogen stress induction [NSI]). In Nostoc punctiforme, they are also induced by the exudate (hormogonium-inducing factor [HIF]) of a symbiotic plant partner. Time course (0.5 to 24 h) transcription profiles were determined by DNA microarray assays for hormogonia of N. punctiforme following induction by HIF and NSI. Clustering analysis revealed both common and distinct transcriptional patterns for the two methods of induction. By 24 h, a common set of 1,328 genes was identified. This 24-h common set of genes arose by the transition of 474 genes from an 819-member common set of genes at 1 h after induction; 405 and 51 genes unique to the HIF and NSI groups at 1 h, respectively; and 398 genes differentially transcribed at later time points. The NSI hormogonia showed a transcriptional checkpoint at 12 h following induction in which up-and downregulated genes were transiently down-or upregulated, respectively. The transient changes in these 1,043 genes appeared to reflect a switch back to a vegetative growth state. Such a checkpoint was not seen in HIF hormogonia. Genes uniquely upregulated in HIF hormogonia included those encoding proteins hypothesized to synthesize a metabolite repressor of hormogonium differentiation. Approximately 34 to 42% of the 6,893 printed genes were differentially transcribed during hormogonium differentiation; about half of those genes were upregulated, and 1,034 genes responded within 0.5 h after induction. These collective results indicate extensive and rapid global changes in the transcription of specific genes during the differentiation of these specialized filaments.

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Phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri catalyzes the nicotinamide adenine dinucleotide-dependent oxidation of phosphite to phosphate. The enzyme belongs to the family of d-hydroxy acid dehydrogenases (DHDHs). A search of the protein databases uncovered many additional putative phosphite dehydrogenases. The genes encoding four diverse candidates were cloned and expressed, and the enzymes were purified and characterized. All oxidized phosphite to phosphate and had similar kinetic parameters despite a low level of pairwise sequence identity (39–72%). A recent crystal structure identified Arg301 as a residue in the active site that has not been investigated previously. Arg301 is fully conserved in the enzymes shown here to be PTDHs, but the residue is not conserved in other DHDHs. Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ∼100-fold decrease in kcat and an almost 700-fold increase in Km,phosphite for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher kcat than the parent PTDH, and a more modest increase in Km for phosphite (nearly 40-fold). Given these results, Arg301 may be involved in the binding and orientation of the phosphite substrate and/or play a catalytic role via electrostatic interactions. Three other residues in the active site region that are conserved in the PTDH orthologs but not DHDHs were identified (Trp134, Tyr139, and Ser295). The importance of these residues was also investigated by site-directed mutagenesis. All of the mutants had kcat values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

The filamentous cyanobacterium Nostoc punctiforme differentiates from vegetative cells into three distinct cell types, heterocysts, hormogonia, and akinetes, in response to different stimuli. Cultures growing with ammonium can be induced to form hormogonia or heterocysts upon removal of the combined nitrogen. A DNA microarray consisting of 94% of the open reading frames predicted from the 9.059-Mb N. punctiforme genome was used to generate a global transcription data set consisting of seven time points over a 24-h period of nitrogen deprivation, which results in heterocyst formation. This data set was compared to a similarly generated data set of nitrogen-starved N. punctiforme resulting in hormogonium formation that had previously been published (E. L. Campbell, H. Christman, and J. C. Meeks, J. Bacteriol. 190:7382-7391, 2008). The transition from vegetative cells to either heterocysts or hormogonia resulted in rapid and sustained expression of genes required for utilization of alternate nitrogen sources. Overall, 1,036 and 1,762 genes were found to be differentially transcribed during the heterocyst and hormogonium time courses, respectively, as analyzed with the Bayesian user-friendly software for analyzing time series microarray experiments (BATS). Successive transcription of heterocyst regulatory, structural, and functional genes occurred over the 24 h required to form a functional heterocyst. During hormogonium differentiation, some heterocyst structural and functional genes were upregulated, while the heterocyst master regulator hetR was downregulated. There are commonalities in differential expression between cells bound for differentiation into heterocysts or hormogonia, yet the two paths are distinguished by their developmentally specific transcription profiles.

Legionella pneumophila replicates in macrophages in a host-derived phagosome, termed the Legionella-containing vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into the macrophage cytosol is well established, the location of type II secretion (T2S) substrates in the infected host cell is unknown. Here, we show that the T2S substrate ProA, a metalloprotease, translocates into the cytosol of human macrophages, where it associates with the LCV membrane (LCVM). Translocation is detected as early as 10 h postinoculation (p.i.), which is approximately the midpoint of the intracellular life cycle. However, it is detected as early as 6 h p.i. if ProA is hyperexpressed, indicating that translocation depends on the timing of ProA expression and that any other factors necessary for translocation are in place by that time point. Translocation occurs with all L. pneumophila strains tested and in amoebae, natural hosts for L. pneumophila. It was absent in murine bone marrow-derived macrophages and murine macrophage cell lines. The ChiA chitinase also associated with the cytoplasmic face of the LCVM at 6 h p.i. and in a T2S-dependent manner. Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced by damage to host membranes, appeared within the LCV of infected human but not murine macrophages beginning at 6 h p.i. Thus, we hypothesize that ProA and ChiA are first secreted into the vacuolar lumen by the activity of the T2S and subsequently traffic into the macrophage cytosol via a novel mechanism that involves a semipermeable LCVM.

Most Salmonella serovars are general pathogens that infect a variety of hosts. These “generalist” serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.