Search

Abstract Background All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA). Results Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns. Conclusion The utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.

The histidine (his) biosynthetic pathway is the most studied biochemical pathway since the acceptance of the operon theory. The his pathway has been shown to be present from single cell forms to complex, multi-cell forms. The his genes have been cloned and sequenced in more than 20 species and have been shown to be controlled posttranscriptionally by a system of segmental stabilization of regions in the transcription code.

The eight Escherichia coli enzymes involved in the synthesis of histidine are encoded by the hisGDCBHAFI operon. Based on sequence data previously established, different restriction sites were deduced and used to map the position of the operon within the Kohara physical map. The results showed that the histidine operon is located around kb 2,100, corresponding to the 44-minute region.

In bacteria, conditions that uncouple translation from transcription activate intragenic terminators located within cistrons. We analyzed the function of NusA in intragenic termination, making use of two tandem terminators located within the hisG cistron, GTTE1 and GTTE2. GTTE2 is a canonical Rho site, capable to terminate with Rho alone in vitro. By contrast, GTTE1 is a suboptimal terminator, featuring a boxA element and requiring a functional NusB to terminate efficiently in vivo. We found that a functional NusA is necessary for efficient termination events to occur at both GTTE1 and 2. To enhance termination at GTTE1 in conditions in which the transcript is free of ribosomes, NusA acts at the same step as NusB and NusE/S10. In the presence of concomitant translation, termination at GTTE1 is dependent on the relative position of the translation stop codon and boxA. If translation stops upstream of boxA, NusA acts at the same step as NusB enhance termination. Ribosomes terminating translation at boxA influence termination at GTTE1. Interactions of NusA and/or NusB with ribosomal components, including NusE/S10, might facilitate termination. Differently from what observed at GTTE1, the NusA-stimulated pausing seems to be sufficient for the occurrence of complete termination events at GTTE2. A functional NusA is also necessary to prevent premature termination of normally translated transcripts. Our data support the hypothesis that NusA may program a fraction of the RNA polymerase to terminate transcription upon interactions with specific sites on the nascent mRNA and either other Nuses or ribosomes. [Copyright &y& Elsevier]