An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola

• Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the gibberellin (GA) phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid (JA) mediated defense response. • Here the function of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae investigated in over 100 isolates. • The Xoc operon leads to production of the bioactive GA4, an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (\u3e90%), but absent in the other major oryzae pathovar. • These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice

By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version — whether defined at the sequence, structure or codon usage level — does not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification

Bacterial strains and plasmids used in this study.

a<p><i>E. coli</i> host for the <i>rnpA</i> knockout protocol; acquired from <i>E. coli</i> Genetic Stock Center, Yale, USA.</p>b<p>used for amplification of the <i>rnpA</i> gene.</p>c<p>template for the amplification of the antibiotic cassette for the gene inactivation protocol; provided by John Roth, University of California, Davis, USA.</p>d<p>template for the <i>gfp</i> amplification.</p>e<p>Heterologous complementation plasmids.</p

Growth curves of test lineages.

<p>Growth curves for each of the test lineages averaged over >20 replicates (solid lines) are compared to the control lineage (5 replicates) (dashed lines). Control lineage curves represent the growth of <i>E. coli</i> MTea1 expressing the wild-type <i>rnpA</i> in pSWAP (MTea1/pSWAP-Ec). The test lineages depicted harbor the following heterologous <i>rnpA</i>s in an MTea1 background: A) <i>P. mirabilis</i> (Pm), B) <i>P. aeruginosa</i> (Pa), C) <i>A. baumannii</i> (Ab), D) <i>N. gonorrhoeae</i> (Ng), E) <i>T. maritima</i> (Tm), F) <i>S. aureus</i> (Sa), G) <i>B. subtilis</i> (Bs), H) <i>S. oralis</i> (So).</p

Average growth parameters (µmax and TTI) from growth curves.

a<p>Relative µmax means the ratio between the average µmax of each test lineage and the average µmax of the control lineage MTea1/pSWAP-Ec.</p>b<p>Relative TTI means the ratio between the average TTI of each test lineage and the average TTI of the control lineage MTea1/pSWAP-Ec.</p

Effect of heterolog divergence on growth parameters of test lineages.

<p>A) Relative maximum specific growth rates (normalized to MTea1/pSWAP-Ec) for each test lineage. µmax is defined as the maximum growth rate in the Richards growth model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032456#pone.0032456-Richards1" target="_blank">[23]</a>. B) Differences in the time to inflection of each test lineage relative to MTea1/pSWAP-Ec. The time to inflection (TTI) is defined as the length of time required to reach µmax. Values for the control lineage are arbitrarily set to 1. Asterisks indicate p values <0.001.</p

Correlation between growth parameters of test lineages and the Codon Adaptation Index (CAI).

<p>Solid symbols represent the values for <i>E. coli</i>; open symbols represent heterologous <i>rnpA</i> CAI and growth parameters of the test lineages, labeled as follows: Ab, <i>A. baumannii</i>; Bs, <i>B. subtilis</i>; Ec, <i>E. coli</i>; Pa, <i>P. aeruginosa</i>; Pm, <i>P. mirabilis</i>; Ng, <i>N. gonorrhoeae</i>; Sa, <i>S. aureus</i>; So, <i>S. oralis</i>; Tm, <i>T. maritima</i>.</p

Correlation between <i>rnpA</i> divergence and growth parameters in test lineages.

<p>Panels A) and B) show the relationship between the previously defined growth parameters (µmax and TTI, respectively) and the corrected per-site divergence (number of amino-acid substitutions per amino-acid site using JTT substitution model as implemented in MEGA5.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032456#pone.0032456-Tamura1" target="_blank">[51]</a>). The dotted line represents growth parameter values for the <i>E. coli</i> control lineage, arbitrarily set to 1. Symbols are labeled to indicate the source organism of the <i>rnpA</i> heterolog present in pSWAP: Ab, <i>A. baumannii</i>; Bs, <i>B. subtilis</i>; Ec, <i>E. coli</i>; Pa, <i>P. aeruginosa</i>; Pm, <i>P. mirabilis</i>; Ng, <i>N. gonorrhoeae</i>; Sa, <i>S. aureus</i>; So, <i>S. oralis</i>; Tm, <i>T. maritima</i>. Solid line represents best-fit linear regression.</p

Phylogenetic range of <i>rnpA</i> heterologs investigated in this study.

<p>The phylogenetic position and range of sources of heterologous <i>rnpA</i> cloned into pSWAP for complementation and fitness studies are shown on a consensus eubacterial phylogenetic tree. The sampled branches are shown in boldface, and the species listed in italics alongside. <i>Escherichia coli</i> (bold) was used as control. The two numbers in the parentheses quantify the pairwise divergence between the heterologous and the <i>E. coli rnpA</i> sequences: the first number is the unadjusted pairwise amino-acid divergence across the entire length of the sequence (expressed as a percentage), the second the corrected per-site divergence (number of amino-acid substitutions per amino-acid site using JTT substitution model as implemented in MEGA5.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032456#pone.0032456-Tamura1" target="_blank">[51]</a>). Eubacterial phylogenetic tree adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032456#pone.0032456-Woese1" target="_blank">[47]</a>.</p