The cell envelope is the target for many antibiotics. In Gram-positive bacteria, membrane alterations and dysfunction caused by antibiotics are sensed mainly by two classes of signal transduction systems: the ECF sigma factors and the two-component signal transduction systems (TCSs). Enduracidin is an antibiotic that inhibits the transglycosylation step of peptidoglycan biosynthesis, and is an attractive target for further antibiotic development studies. We assessed transcriptional responses to enduracidin in Bacillus subtilis cells using a high-density tiling chip, and compared the results with responses to bacitracin, which inhibits the lipid II cycle of peptidoglycan synthesis. We exploited the quantitative advantage of the tiling chip to introduce a new criterion, an increase in transcriptional level, in addition to the conventional induction ratio, in order to distinguish genes of biological significance from those with lower induction ratios. Our results indicate that introduction of the new criterion led to unambiguous identification of core transcriptional responses to antibiotics, with a reduction in the number of possible background genes, compared to previous results obtained using gene arrays. We identified 129 genes that were significantly upregulated by enduracidin and/or bacitracin. Notably, we found that inactivation of the LiaRS TCS, which was the system most strongly induced by the two antibiotics, resulted in increased sensitivity to enduracidin, probably through a failure to induce LiaIH proteins. We noted that 33 genes belonging to the SigM regulon were induced by both antibiotics. Consistent with stronger induction of the SigM regulon in enduracidin-treated cells, inactivation of sigM resulted in increased sensitivity to enduracidin. In addition, and for the first time, we found that the Spx regulon was induced in cells challenged by enduracidin and bacitracin, suggesting that thiol-oxidative stress occurred in cells treated with antibiotics. These findings contribute to further our understanding of the molecular nature of genetic systems involved in antibiotic resistance.

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The cell wall integrity checkpoint coordinates cell wall synthesis and mitosis in the budding yeast, Saccharomyces cerevisiae. It has been reported that this checkpoint arrests the cell cycle at G2/M phase with repression of the M phase cyclin Clb2p at the transcriptional level, under perturbation of cell wall synthesis. We demonstrate that an override of this checkpoint with accumulation of CLB2 mRNA is induced when negative CLB2 transcription factors are deleted or when positive CLB2 transcription factors are overproduced in cell wall-defective cells. Our data imply that transcription factors for CLB2 are involved in the cell wall integrity checkpoint system and suggest that there are multiple regulation pathways of the checkpoint.

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In mangrove species the past geomorphic changes in coastal regions and reproductive systems are important factors of their distribution and genetic structure of populations. However, very little is known about the levels of genetic variation of Rhiozophora species in Southeast Asia. In this study, we surveyed levels and patterns of genetic variation as well as population structure of two sympatric mangrove species, Rhizophora apiculata and R. mucronata in Thailand, using five nuclear genes and two cpDNA regions. In all investigated DNA regions, nucleotide variation within species was low, while nucleotide divergence between the two species was considerable. The nuclear genes evolved 10 times faster than the cpDNA regions. In both R. apiculata and R. mucronata, significant positive F_IS values were found, indicating deviation from Hardy-Weinberg proportions and a deficiency of heterozygotes. In both species, we found significant genetic differentiation between populations. However, the pattern of population differentiation (F_ST) of R. apiculata differed from that of R. mucronata. Our results suggest that the two investigated species have different demographic history, even though they are sympatric and have similar reproductive systems.

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The effect of geographical isolation on speciation, particularly within short geographical ranges, is poorly understood among marine organisms. Focusing on marine lakes of the Palau Islands, we investigated the effect of geographical isolation on Sphaeramia orbicularis, a coastal fish inhabiting marine lakes and lagoons. We collected a total of 157 individuals from three meromictic marine lakes and three lagoon sites, and analyzed the genetic diversity and differentiation of the populations based on complete sequences of the mitochondrial control region (824 bp). The analyses show that the genetic diversity of marine lake populations is much lower than that of lagoon populations. Moreover, a mismatch distribution analysis suggests that marine lake populations have experienced a decrease followed by a rapid expansion of their population size. These results reveal that marine lake populations have experienced severe founder and/or bottleneck events during the last thousand to tens of thousand years. Pairwise Φ_ST values ranged from 0.531 to 0.848 between marine lake and lagoon populations and from 0.429 to 0.870 among marine lake populations, indicating a high degree of genetic differentiation. We speculate that such peripatric differentiation between marine lake and lagoon populations was caused by a small number of individuals colonizing the lakes from the lagoon (founder event) followed by repetitive bottleneck events, such as those generated by the El Niño-Southern Oscillation (ENSO). So far, such high genetic divergences in extremely short geographical ranges (approximately 150–250 m) have scarcely been reported for marine organisms. We suggest that the marine lake is one of the good model of geographical isolation in marine organisms and each marine lake population is in the early stages of speciation.

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Mitochondrial (mt) heteroplasmy in the control region (CR) of the black-faced spoonbill was investigated using LA-PCR. To avoid amplification of transpositioned nuclear genome fragment from mtDNA (numt), PCR product of the almost-complete mitochondrial genome was amplified using primers designed to anneal on the COIII gene. Then nested LA-PCR product was amplified between the cyt b and 12S rRNA genes using the almost-complete mitochondrial genome PCR product as a template. Nucleotide sequencing revealed tandem duplication composed of two units. The first contains cyt b-1, tRNA^Thr-1, tRNA^Pro-1, ND6-1, tRNA^Glu-1 and CR1, and the second consists of cyt b-2, tRNA^Thr-2, tRNA^Pro-2, ND6-2, tRNA^Glu-2 and CR2, followed by tRNA^Phe and 12S rRNA. The duplicated cyt b-2 sequence coincided with 499 bp at the 3’ end of cyt b-1. With the exception of the CR, the other genes in the duplicated sequence were identical to the original corresponding gene. Even though both CR1 and CR2 contain functional blocks, such as a poly-C site, a goose hairpin and a TAS structure in Domain I, the 3’ end of CR1 was followed by a 112 bp sequence (non-coding region) that was not found in CR2 or in sequence homology analysis of similar genes. Meanwhile, CR2 ended in a complicated repeat sequence. The 5’ franking region in the Domain I (Region A) and the 3’ franking region in the Domain I (Region B) of the two CRs evolve in quite different manners: Region A was highly variable between CR1 and CR2 in the same individuals, while Region B was almost identical between them, which indicates concerted evolution.

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Identifying loci under natural selection from genomic surveys is of great interest in different research areas, stimulated by the increasing ease with large numbers of markers to gain a genome-wide perspective on differentiation. In this study, we searched for the genetic signatures of selection by screening 114 amplified fragment length polymorphism (AFLP) markers in three finless porpoise populations inhabiting contrasting natural environments (freshwater and marine habitat). Comparing among three populations, four AFLP loci exhibited F_ST values higher than 0.975 quantile which might be inferred to be under divergent selection and two loci fell below the 0.025 quantile which might be affected by balancing selection. Although these loci were not supported with statistical significance in false discovery rate (FDR) analysis, the present study illustrated the potential of genome-wide surveys to identify specific genome regions or genes associated with freshwater adaptation of the finless porpoise.

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A genetic tool to introduce marker-free deletions is essential for multiple manipulations of genomes. We report a simple and efficient method to create marker-free deletion mutants of Bacillus subtilis through transformation with recombinant PCR products, using the Escherichia coli mazF gene encoding an endoribonuclease that cleaves free mRNAs as a counter-selection tool. Our method will be applicable to any bacterium in which introduction of the mazF cassette into the genome by double crossover homologous recombination is possible.

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