Recent advanced studies of genomics and proteomics have revealed the variation and diversity of ribosomal proteins (r-proteins) in different organisms and organelles. Radical free and highly reducing (RFHR) two-dimensional (2-D) electrophoresis is known to be powerful for separating ribosomal proteins that are usually small and basic, and not separated well by standard 2-D electrophoresis. Using the RFHR method, we investigated the protein profile of the Bacillus subtilis ribosomes by a proteomic approach. We found that two L31 paralogue proteins (RpmE and YtiA) showed different temporal expression patterns in the ribosomes. The RpmE protein, which is an L31 variant with a Zn-binding motif, binds one zinc ion at the motif, which is required for stabilization of the protein in the cell. On the other hand, the expression of the ytiA gene, which encodes another L31 variant (YtiA) without the Zn-binding motif, is negatively controlled by the zinc-specific transcriptional repressor Zur and is likely induced by zinc starvation. This article reviews the recent findings that replacement of two types of L31 proteins in the ribosome is controlled by the intracellular zinc concentration.
The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.
The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65°C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65°C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.
This study was focused on obtaining the complete gene sequence of the toxR gene in V. harveyi by using toxR-targeted PCR to amplify 5′ and 3′ regions flanking the 576-bp Vibrio harveyi (NBRC 15634) toxR gene fragment previously amplified using degenerate PCR. To obtain the 5′ flanking sequences, a forward PCR primer (VhtoxRpv) was designed based on known sequences upstream of toxR in V. parahaemolyticus and V. vulnificus. The reverse primer (VctoxR2R) was based on the sequence of the 576-bp Vibrio harveyi toxR fragment. The resulting 750-bp amplicon was sequenced, providing the 5′ sequences of the V. harveyi (NBRC 15634) toxR gene. The 3′ flanking region was amplified using a primer pair toxRS1 and toxRS2 based on V. parahaemolyticus and V. vulnificus toxR and toxS, resulting in a 900-bp amplicon that contained the remaining 3′ sequences of the V. harveyi NBRC 15634 toxR. This paper reports, for the first time, a complete 882-bp nucleotide sequence for toxR in Vibrio harveyi. Sequence analysis and alignment revealed that the complete toxR gene in V. harveyi shares 87% sequence similarity with toxR of V. parahaemolyticus, 84% similarity with V. fluvialis, 83% with V. vulnificus and partial sequence of V. campbellii. The phylogenetic trees revealed wider divergence in toxR compared to 16S rRNA genes, so that V. harveyi could easily be distinguished from V. campbellii and V. parahaemolyticus.