Biomedical subjects
Stanley Maloy
Publications and source records attributed to Stanley Maloy.
Facile approach for constructing TEV insertions to probe protein structure in vivo.
The tobacco etch virus (TEV) protease has been used as a tool to examine protein structure in vivo. TEV cleavage sites (TEVcs) have been introduced via cloning into unique restriction sites or random transposon mutagenesis. We describe a facile, efficient method for introducing TEVcs at precise locations in a gene to test specific predictions about protein structure. The method uses the lamda Red recombination system to construct seamless, in-frame insertions of the TEVcs at any desired location within an open reading frame (ORF). The system was tested using the multifunctional PutA protein Salmonella enterica sv. Typhimurium. The first step involved insertion of a chloramphenicol resistance (Cam(R)) cassette with a transcriptional terminator at the desired location. A second swap then replaces the Cam(R) insertion with the TEVcs. Placing a copy of the lac operon downstream of the putA gene provides a simple counterselection for replacement of the Cam(R) insertion and also provides a reporter gene for monitoring transcription of the mutated gene.
The era of microbiology: a golden phoenix.
The discoveries over the last decade have demonstrated that microbiology is a central scientific discipline with practical applications in agriculture, medicine, bioremediation, biotechnology, engineering, and other fields. It is clear that the roles of microbes in nature are so diverse that the process of mining this genetic variation for new applications will continue long into the future. Moreover, the rapid rate of microbial evolution ensures that there will be no permanent solution to agricultural, medical, or environmental problems caused by microbes. These problems will demand a continual stream of creative new approaches that evolve along with the microbes. Thus, the excitement of this field will continue long into the future. However, these opportunities and imperatives demand a deep understanding of basic microbial physiology, genetics, and ecology. Major challenges that lay ahead are to impart the broad training needed to entice and enable the next generation of microbiologists, and to educate the public and government representatives about the continued and critical importance of this field for health and the economy.
Pigeon-associated strains of Salmonella enterica serovar Typhimurium phage type DT2 have genomic rearrangements at rRNA operons.
Strains from a subgroup of Salmonella enterica serovar Typhimurium frequently associated with pigeon infections were tested for genomic anomalies and virulence in mice. Some strains have a genomic inversion between rrn operons. Two prophages found in the common laboratory strain LT2 were absent. Pigeon-associated strains are still virulent in mice.
Genomic rearrangements at rrn operons in Salmonella.
Most Salmonella serovars are general pathogens that infect a variety of hosts. These "generalist" serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.
A cryptic proline permease in Salmonella typhimurium.
Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy done of the proY+ gene or both a single copy of the proY+ gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.