Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas.

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc.

The turnover of strains in intermittent and persistent nasal carriers of Staphylococcus aureus.

OBJECTIVES: We aimed to examine the dynamics of Staphylococcus aureus nasal carriage in healthy adults. METHOD: Selected S. aureus strains isolated from weekly nasal swabs obtained from 122 healthy young adults over a 13 week period were spa typed. RESULTS: The median duration of intermittent carriage was 4 weeks (IQR 2-6) and the median interval between episodes of carriage of different spa types was 3.5 weeks (IQR 2.25-4). 6/19 (32%) Persistent carriers were colonised with more than one spa type during the study, and in two persistent carriers a brief period of mixed colonisation with two spa types was observed. Even when the carriage strain changed, it was very rare for persistent carriers to have a period during which they were culture-negative (only 6/188 (3%) swabs submitted by persistent carriers failed to culture S. aureus). CONCLUSIONS: Our results imply that at least every eight weeks a healthy young adult is exposed to S. aureus sufficient to cause a new episode of carriage among intermittent carriers. Persistent carriers are almost always colonised with S. aureus and over the course of a year there will be at least one replacement of the dominant strain.

Testing temperature-induced proteomic changes in the plant-associated bacterium Pseudomonas fluorescens SBW25.

Traits used by bacteria to enhance ecological performance in natural environments are not well understood. Recognizing that the saprophytic plant-colonizing bacterium Pseudomonas fluorescens SBW25 experiences temperatures in its natural environment significantly cooler than the 28°C routinely used in the laboratory, we identified proteins differentially expressed between 28°C and the more environmentally relevant temperature of 14°C. Of 2102 protein isoforms, 32 were temperature responsive and identified by mass spectrometry. Seven of these (OmpR, MucD, GuaD, OsmY and three of unknown function, Tee1, Tee2 and Tee3) were selected for genetic and ecological analyses. In each instance, changes in protein expression with temperature were mirrored by parallel transcriptional changes. The fitness contribution of the genes encoding each of the seven proteins was larger at 14°C than 28°C and included two cases of trade-offs (enhanced fitness at one temperature and reduced fitness at the other -mucD and tee2 deletions). The relationship between the fitness effects of genes in vitro and in vivo was variable, but two temperature-responsive genes -osmY and mucD- contribute substantially to the ability of P. fluorescens to colonize the plant environment.

The structure-function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain.

The GGDEF response regulator WspR couples the chemosensory Wsp pathway to the overproduction of acetylated cellulose and cell attachment in the Pseudomonas fluorescens SBW25 wrinkly spreader (WS) genotype. Here, it is shown that WspR is a diguanylate cyclase (DGC), and that DGC activity is elevated in the WS genotype compared to that in the ancestral smooth (SM) genotype. A structure-function analysis of 120 wspR mutant alleles was employed to gain insight into the regulation and activity of WspR. Firstly, 44 random and defined pentapeptide insertions were produced in WspR, and the effects determined using assays based on colony morphology, attachment to surfaces and cellulose production. The effects of mutations within WspR were interpreted using a homology model, based on the crystal structure of Caulobacter crescentus PleD. Mutational analyses indicated that WspR activation occurs as a result of disruption of the interdomain interface, leading to the release of effector-domain repression by the N-terminal receiver domain. Quantification of attachment and cellulose production raised significant questions concerning the mechanisms of WspR function. The conserved RYGGEEF motif of WspR was also subjected to mutational analysis, and 76 single amino acid residue substitutions were tested for their effects on WspR function. The RYGGEEF motif of WspR is functionally conserved, with almost every mutation abolishing function.

Experimental adaptation to high and low quality environments under different scales of temporal variation.

We investigated the role of the scale of temporal variation in the evolution of generalism in populations of the bacterium Pseudomonas fluorescens. Replicate populations were propagated as batch cultures for approximately 1400 generations (192 days), in either high quality media only, low quality media only, or were alternated between the two at a range of temporal scales (between 1 and 48 days). Populations evolved in alternating media showed fitness increases in both media and the rate of alternation during selection had no effect on average fitness in either media. Moreover, the fitness of these populations in high quality media was the same as for populations evolved only in high quality media and likewise for low quality media. Populations evolved only in high or low quality media did not show fitness improvements in their nonselective media. These results indicate that cost-free generalists can evolve under a wide range of temporal variation.

Spatial heterogeneity and the stability of host-parasite coexistence.

Spatially heterogeneous environments can theoretically promote more stable coexistence of hosts and parasites by reducing the risk of parasite attack either through providing permanent spatial refuges or through providing ephemeral refuges by reducing dispersal. In experimental populations of Pseudomonas aeruginosa and the bacteriophage PP7, spatial heterogeneity promoted stable coexistence of host and parasite, while coexistence was significantly less stable in the homogeneous environment. Phage populations were found to be persisting on subpopulations of sensitive bacteria. Transferring populations to fresh microcosms every 24 h prevented the development of permanent spatial refuges. However, the lower dispersal rates in the heterogeneous environment were found to reduce parasite transmission thereby creating ephemeral refuges from phage attack. These results suggest that spatial heterogeneity can stabilize an otherwise unstable host-parasite interaction even in the absence of permanent spatial refuges.

Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25.

In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.

In vivo expression technology strategies: valuable tools for biotechnology.

Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function. It is probable that many of these are required for survival in environments other than the agar plate. In vivo selection strategies provide a means of obtaining genes active in complex natural environments. Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology.

Diversity peaks at intermediate productivity in a laboratory microcosm.

The species diversity of natural communities is often strongly related to their productivity. The pattern of this relationship seems to vary: diversity is known to increase monotonically with productivity, to decrease monotonically with productivity, and to be unimodally related to productivity, with maximum diversity occurring at intermediate levels of productivity. The mechanism underlying these patterns remains obscure, although many possibilities have been suggested. Here we outline a simple mechanism--involving selection in a heterogeneous environment--to explain these patterns, and test it using laboratory cultures of the bacterium Pseudomonas fluorescens. We grew diverse cultures over a wide range of nutrient concentrations, and found a strongly unimodal relationship between diversity and productivity in heterogeneous, but not in homogeneous, environments. Our result provides experimental evidence that the unimodal relationship often observed in natural communities can be caused by selection for specialized types in a heterogeneous environment.

Disturbance and diversity in experimental microcosms.

External agents of mortality (disturbances) occur over a wide range of scales of space and time, and are believed to have large effects on species diversity. The "intermediate disturbance hypothesis", which proposes maximum diversity at intermediate frequencies of disturbance, has received support from both field and laboratory studies. Coexistence of species at intermediate frequencies of disturbance is thought to require trade-offs between competitive ability and disturbance tolerance, and a metapopulation structure, with disturbance affecting only a few patches at any given time. However, a unimodal relationship can also be generated by global disturbances that affect all patches simultaneously, provided that the environment contains spatial niches to which different species are adapted. Here we report the results of tests of this model using both isogenic and diverse populations of the bacterium Pseudomonas fluorescens. In both cases, a unimodal relationship between diversity and disturbance frequency was generated in heterogeneous, but not in homogeneous, environments. The cause of this relationship is competition among niche-specialist genotypes, which maintains diversity at intermediate disturbance, but not at high or low disturbance. Our results show that disturbance can modulate the effect of spatial heterogeneity on biological diversity in natural environments.

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

Evolutionary genetics: The economics of mutation.

The presence of mutator genotypes in populations of bacteria may be favoured by selection because they produce rare beneficial mutations and thereby increase the rate of adaptive evolution. Recent work, however, shows that the relationship between mutation rates and adaptive evolution is more complicated.

Adaptation of Pseudomonas fluorescens to the plant rhizosphere.

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced (rhi) genes have homology to known non-Pseudomonas genes. One of the rhi genes (hrcC) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.

Detecting linkage disequilibrium in bacterial populations.

The distribution of the number of pairwise differences calculated from comparisons between n haploid genomes has frequently been used as a starting point for testing the hypothesis of linkage equilibrium. For this purpose the variance of the pairwise differences, VD, is used as a test statistic to evaluate the null hypothesis that all loci are in linkage equilibrium. The problem is to determine the critical value of the distribution of VD. This critical value can be estimated either by Monte Carlo simulation or by assuming that VD is distributed normally and calculating a one-tailed 95% critical value for VD, L, L = EVD + 1.645 sqrt(VarVD), where E(VD) is the expectation of VD, and Var(VD) is the variance of VD. If VD (observed) > L, the null hypothesis of linkage equilibrium is rejected. Using Monte Carlo simulation we show that the formula currently available for Var(VD) is incorrect, especially for genetically highly diverse data. This has implications for hypothesis testing in bacterial populations, which are often genetically highly diverse. For this reason we derive a new, exact formula for Var(VD). The distribution of VD is examined and shown to approach normality as the sample size increases. This makes the new formula a useful tool in the investigation of large data sets, where testing for linkage using Monte Carlo simulation can be very time consuming. Application of the new formula, in conjunction with Monte Carlo simulation, to populations of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Bacillus subtilis reveals linkage disequilibrium where linkage equilibrium has previously been reported.

Adaptive radiation in a heterogeneous environment.

Successive adaptive radiations have played a pivotal role in the evolution of biological diversity. The effects of adaptive radiation are often seen, but the underlying causes are difficult to disentangle and remain unclear. Here we examine directly the role of ecological opportunity and competition in driving genetic diversification. We use the common aerobic bacterium Pseudomonas fluorescens, which evolves rapidly under novel environmental conditions to generate a large repertoire of mutants. When provided with ecological opportunity (afforded by spatial structure), identical populations diversify morphologically, but when ecological opportunity is restricted there is no such divergence. In spatially structured environments, the evolution of variant morphs follows a predictable sequence and we show that competition among the newly evolved niche-specialists maintains this variation. These results demonstrate that the elementary processes of mutation and selection alone are sufficient to promote rapid proliferation of new designs and support the theory that trade-offs in competitive ability drive adaptive radiation.

Bacterial genomics and adaptation to life on plants: implications for the evolution of pathogenicity and symbiosis.

Many bacteria form intimate associations with plants. Despite the agricultural and biotechnological significance of these bacteria, no whole genome sequences have yet been described. Plant-associated bacteria form a phylogenetically diverse group, with representative species from many major taxons. Sequence information from genomes of closely related bacteria, in combination with technological developments in the field of functional genomics, provides new opportunities for determining the origin and evolution of traits that contribute to bacterial fitness and interactions with plant hosts.

Single-step conjugative cloning of bacterial gene fusions involved in microbe-host interactions.

In vivo expression technology (IVET) is a genetic strategy for isolating genes expressed in vivo. In order to full exploit this technology, it is necessary to analyse large numbers of IVET-generated gene fusions, which must be recovered from the chromosome of host bacteria. In bacteria for which transductional methods are not available, the recovery of integrated fusion plasmids is problematic and currently limits broad application of IVET. We describe a rapid, single-step, triparental conjugative approach for recovering chromosomally integrated fusion plasmids from both Pseudomonas fluorescens and Salmonella typhimurium. This simple and broadly applicable conjugative cloning system extends the utility of the IVET approach to clinically and agronomically relevant microbes and may be employed to recover non-replicating and integrated plasmids in other systems.

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome.

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 PacI, 53 SpeI, 71 XbaI) were placed on the physical map and complete maps of the circular chromosome were obtained for both PacI and SpeI; only XbaI fragments linking SpeI fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by southern hybridization and 31 genes were positioned including oriC, rDNA operons (rnnA-E), recA, gacA, and pyvD.