CelR, an ortholog of the diguanylate cyclase PleD of Caulobacter, regulates cellulose synthesis in Agrobacterium tumefaciens.

Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens. In this study, we identified two putative diguanylate cyclase genes, celR (atu1297) and atu1060, that influence production of cellulose in A. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion of celR, but not atu1060, resulted in decreased cellulose biosynthesis. celR overexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis of celR and Δcel mutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing both celR and celA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lack celA use CelR, called PleD, to regulate specific processes associated with polar localization and cell division.

Do it right, not over: hidden secrets to insurance efficiency.

If you took a poll of all the dental offices across the United States, their number one frustration would be the challenge of dealing with insurance claims. Dental benefits plans can have limited payment amounts, claims can get delayed or lost in the processing, and claims reviewers will often lower payment on a dental treatment by paying an alternate benefit. Filing claims and receiving reimbursement for them has remained a frustration for decades. With dental plans numbering in the thousands, there are countless confusing rules and regulations in the processing; there are exclusions, alternate benefit guidelines, specific policies, frequency limitations, and complicated contact information, to name a few. Needless to say many mistakes are made in filing these dental claims, causing the need for multiple submissions. This, in turn, can cause delays, resulting in an out-of-control accounts receivable situation. Dentistry has, for years, allowed patients to depend on them 100 percent for the accuracy of their dental benefits and the outcome of their dental claims. This should be a shared responsibility.

An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect.

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria.

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

Unmasking host and microbial strategies in the Agrobacterium-plant defense tango.

Coevolutionary forces drive adaptation of both plant-associated microbes and their hosts. Eloquently captured in the Red Queen Hypothesis, the complexity of each plant-pathogen relationship reflects escalating adversarial strategies, but also external biotic and abiotic pressures on both partners. Innate immune responses are triggered by highly conserved pathogen-associated molecular patterns, or PAMPs, that are harbingers of microbial presence. Upon cell surface receptor-mediated recognition of these pathogen-derived molecules, host plants mount a variety of physiological responses to limit pathogen survival and/or invasion. Successful pathogens often rely on secretion systems to translocate host-modulating effectors that subvert plant defenses, thereby increasing virulence. Host plants, in turn, have evolved to recognize these effectors, activating what has typically been characterized as a pathogen-specific form of immunity. Recent data support the notion that PAMP-triggered and effector-triggered defenses are complementary facets of a convergent, albeit differentially regulated, set of immune responses. This review highlights the key players in the plant's recognition and signal transduction pathways, with a focus on the aspects that may limit Agrobacterium tumefaciens infection and the ways it might overcome those defenses. Recent advances in the field include a growing appreciation for the contributions of cytoskeletal dynamics and membrane trafficking to the regulation of these exquisitely tuned defenses. Pathogen counter-defenses frequently manipulate the interwoven hormonal pathways that mediate host responses. Emerging systems-level analyses include host physiological factors such as circadian cycling. The existing literature indicates that varying or even conflicting results from different labs may well be attributable to environmental factors including time of day of infection, temperature, and/or developmental stage of the host plant.

16S rRNA gene survey of microbial communities in Winogradsky columns.

A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities.

Using comparative genomics for inquiry-based learning to dissect virulence of Escherichia coli O157:H7 and Yersinia pestis.

Genomics and bioinformatics are topics of increasing interest in undergraduate biological science curricula. Many existing exercises focus on gene annotation and analysis of a single genome. In this paper, we present two educational modules designed to enable students to learn and apply fundamental concepts in comparative genomics using examples related to bacterial pathogenesis. Students first examine alignments of genomes of Escherichia coli O157:H7 strains isolated from three food-poisoning outbreaks using the multiple-genome alignment tool Mauve. Students investigate conservation of virulence factors using the Mauve viewer and by browsing annotations available at the A Systematic Annotation Package for Community Analysis of Genomes database. In the second module, students use an alignment of five Yersinia pestis genomes to analyze single-nucleotide polymorphisms of three genes to classify strains into biovar groups. Students are then given sequences of bacterial DNA amplified from the teeth of corpses from the first and second pandemics of the bubonic plague and asked to classify these new samples. Learning-assessment results reveal student improvement in self-efficacy and content knowledge, as well as students' ability to use BLAST to identify genomic islands and conduct analyses of virulence factors from E. coli O157:H7 or Y. pestis. Each of these educational modules offers educators new ready-to-implement resources for integrating comparative genomic topics into their curricula.