The Tandem Repeats Enabling Reversible Switching between the Two Phases of β-Lactamase Substrate Spectrum

Yi, Hyojeong,Song, Han,Hwang, Junghyun,Kim, Karan,Nierman, William C.,Kim, Heenam Stanley Public Library of Science 2014 PLoS genetics Vol.10 No.9

<P>Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (<I>de novo</I> TRs) that occurred in the coding region of a β-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic. Under selective pressure with one of the original substrates (amoxicillin), a high level of reversion occurred in the mutant β-lactamase genes completing a cycle back to the original substrate spectrum. The <I>de novo</I> TRs coupled with reversion makes a genetic toggling mechanism enabling reversible switching between the two phases of the substrate spectrum of β-lactamases. This toggle exemplifies the effective adaptation of <I>de novo</I> TRs for enhanced bacterial survival. We found pairs of direct repeats that mediated the DNA duplication (TR formation). In addition, we found different duos of sequences that mediated the DNA duplication. These novel elements—that we named SCSs (same-strand complementary sequences)—were also found associated with β-lactamase TR mutations from clinical isolates. Both direct repeats and SCSs had a high correlation with TRs in diverse bacterial genomes throughout the major phylogenetic lineages, suggesting that they comprise a fundamental mechanism shaping the bacterial evolution.</P><P><B>Author Summary</B></P><P>β-lactamases can adapt to new antibiotics by mutations in their genes. The original and the extended substrate spectrums of β-lactamases define two phases of catalytic activity, and the conversion by point mutations is unidirectional from the initial to the new spectrum. We describe duplication mutations that enable reversible switching between the substrate spectrums, increasing the adaptability of the bacterium. We provide evidence supporting that two distinct groups of short sequences mediated the formation of DNA duplications in β-lactamases: direct repeats and novel elements that we named, SCSs (same-strand complementary sequences). Our study suggests that DNA duplication processes mediated by both direct repeats and SCSs are not just limited to the β-lactamase genes but comprise a fundamental mechanism in bacterial genome evolution.</P>

The Early Stage of Bacterial Genome-Reductive Evolution in the Host

Song, Han,Hwang, Junghyun,Yi, Hyojeong,Ulrich, Ricky L.,Yu, Yan,Nierman, William C.,Kim, Heenam Stanley Public Library of Science 2010 PLoS pathogens Vol.6 No.5

<▼1><P>The equine-associated obligate pathogen <I>Burkholderia mallei</I> was developed by reductive evolution involving a substantial portion of the genome from <I>Burkholderia pseudomallei</I>, a free-living opportunistic pathogen. With its short history of divergence (∼3.5 myr), <I>B. mallei</I> provides an excellent resource to study the early steps in bacterial genome reductive evolution in the host. By examining 20 genomes of <I>B. mallei</I> and <I>B. pseudomallei</I>, we found that stepwise massive expansion of IS (insertion sequence) elements IS<I>Bma</I>1, IS<I>Bma</I>2, and IS<I>407</I>A occurred during the evolution of <I>B. mallei</I>. Each element proliferated through the sites where its target selection preference was met. Then, IS<I>Bma</I>1 and IS<I>Bma</I>2 contributed to the further spread of IS<I>407</I>A by providing secondary insertion sites. This spread increased genomic deletions and rearrangements, which were predominantly mediated by IS<I>407</I>A. There were also nucleotide-level disruptions in a large number of genes. However, no significant signs of erosion were yet noted in these genes. Intriguingly, all these genomic modifications did not seriously alter the gene expression patterns inherited from <I>B. pseudomallei</I>. This efficient and elaborate genomic transition was enabled largely through the formation of the highly flexible IS-blended genome and the guidance by selective forces in the host. The detailed IS intervention, unveiled for the first time in this study, may represent the key component of a general mechanism for early bacterial evolution in the host.</P></▼1><▼2><P><B>Author Summary</B></P><P>It has been known for some time that bacteria undergo genome-reduction when they transition from a free-living state to a constantly host-restricted state. High levels of IS element expansion were also found in these bacteria, and the IS elements were suggested to play a role in genome reductive evolution. Here we provide evidence for stepwise IS actions as the exclusive mechanism that mediates bacterial genomic changes during the early stage of constant host-bacterial association, by unveiling the processes that resulted in the development of <I>B. mallei</I> genome. We show the details of the multi-level interplay of IS elements, which facilitate the wide spread of the IS copies, and the overall mechanics in genome reduction and rearrangement. These processes appeared to operate as chain reactions mediating elaborate genomic transition, without seriously affecting the original gene expression patterns. The absence of differential gene expression in the resulting genome suggests that changes in transcriptional regulation that are often observed in other old bacterial genomes may take place subsequent to the IS-mediated steps, along with gradual nucleotide-level changes.</P></▼2>

Aflatoxin formation and gene expression in response to carbon source media shift in Aspergillus parasiticus

Wilkinson, J. R.,Yu, J.,Abbas, H. K.,Scheffler, B. E.,Kim, H. S.,Nierman, W. C.,Bhatnagar, D.,Cleveland, T. E. Taylor Francis 2007 Food additives and contaminants Vol.24 No.10

<P> Aflatoxins are toxic and carcinogenic polyketide metabolites produced by fungal species, including Aspergillus flavus and A. parasiticus. The biosynthesis of aflatoxins is modulated by many environmental factors, including the availability of a carbon source. The gene expression profile of A. parasiticus was evaluated during a shift from a medium with low concentration of simple sugars, yeast extract (YE), to a similar medium with sucrose, yeast extract sucrose (YES). Gene expression and aflatoxins (B1, B2, G1, and G2) were quantified from fungal mycelia harvested pre- and post-shifting. When compared with YE media, YES caused temporary reduction of the aflatoxin levels detected at 3-h post-shifting and they remained low well past 12 h post-shift. Aflatoxin levels did not exceed the levels in YE until 24 h post-shift, at which time point a tenfold increase was observed over YE. Microarray analysis comparing the RNA samples from the 48-h YE culture to the YES samples identified a total of 2120 genes that were expressed across all experiments, including most of the aflatoxin biosynthesis genes. One-way analysis of variance (ANOVA) identified 56 genes that were expressed with significant variation across all time points. Three genes responsible for converting norsolorinic acid to averantin were identified among these significantly expressed genes. The potential involvement of these genes in the regulation of aflatoxin biosynthesis is discussed.</P>

Simple Sequence Repeat (SSR)-Based Gene Diversity in Burkholderia pseudomallei and Burkholderia mallei

송한,황정현,Jaehee Myung,Hyoseok Seo,Hyojeong Yi,Hee-Sun Sim,Bong-Su Kim,William C. Nierman,김희남 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.2

Pathogens Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) contain a large number (> 12,000) of Simple Sequence Repeats (SSRs). To study the extent to which these features have contributed to the diversification of genes, we have conducted comparative studies with nineteen genomes of these bacteria. We found 210 genes with characteristic types of SSR variations. SSRs with nonamer repeat units were the most abundant, followed by hexamers and trimers. Amino acids with smaller and nonpolar R-groups are preferred to be encoded by the variant SSRs, perhaps due to their minimal impacts to protein functionality. A majority of these genes appears to code for surface or secreted proteins that may directly interact with the host factors during pathogenesis or other environmental factors. There also are others that encode diverse functions in the cytoplasm, and this protein variability may reflect an extensive involvement of phase variation in survival and adaptation of these pathogens.

Type VI secretion is a major virulence determinant in <i>Burkholderia mallei</i>

Schell, Mark A.,Ulrich, Ricky L.,Ribot, Wilson J.,Brueggemann, Ernst E.,Hines, Harry B.,Chen, Dan,Lipscomb, Lyla,Kim, H. Stanley,Mrá,zek, Jan,Nierman, William C.,DeShazer, David Blackwell Publishing Ltd 2007 Molecular microbiology Vol.64 No.6

<P><B>Summary</B></P><P> <I>Burkholderia mallei</I> is a host‐adapted pathogen and a category B biothreat agent. Although the <I>B. mallei</I> VirAG two‐component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of <I>virAG</I> resulted in transcriptional activation of ∼60 genes, including some involved in capsule production, actin‐based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up‐expressed > 2.5‐fold, but capsule was still produced in the absence of <I>virAG</I>. Actin tail formation required <I>virAG</I> as well as <I>bimB</I>, <I>bimC</I> and <I>bimE</I>, three previously uncharacterized genes that were activated four‐ to 15‐fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up‐expressed as much as 30‐fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS‐PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when <I>virAG</I> was overexpressed. Purified His‐tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp‐family protein is produced <I>in vivo</I> during infection.</P>