Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the six cas genes and one of the two CRISPRs in this region restored biofilm formation and swarming to DMS3 lysogenized strains. Our observations suggest a role for CRISPR regions in modifying the effects of lysogeny on P. aeruginosa.Bacteriophages are probably best known for their role as tools used to study bacteria. Phages have also served as important models for the study of mechanisms of transcription, recombination, and transposition (8). Bacteriophages also shape microbial populations both by impacting the size and structure of bacterial communities and through the transfer of genetic material between bacterial strains (6, 39, 52). It is estimated that phages can lyse as many as 20% of all bacterial cells daily; therefore, these infectious particles can have a profound impact on the evolution of microbes (53).While some bacteriophage infections are primarily lytic, temperate or lysogenic bacteriophages often integrate into the bacterial genome as a prophage causing a chronic infection of the host bacterium (52). In some cases, genes carried by a bacteriophage confer a new function upon a bacterium, typically not directly related to the phage life cycle, through a process known as lysogenic conversion. Lysogenic conversion likely supports phage survival indirectly by increasing the fitness of the host microbe, thus promoting the continued persistence of the phage genome within the host population. Examples of this phenomenon include the phage-mediated introduction of secreted virulence factors such as cholera toxin (30), altered lipopolysaccharide profile (35), and improved adhesion to epithelial cells (50).Here we report that infection of Pseudomonas aeruginosa PA14 by phage DMS3 results in lysogenized strains unable to form a biofilm or undergo swarming motility-two key group behaviors of this bacterium. Furthermore, we show that the loss of biofilm formation and swarming motility requires clustered regular interspaced short palindromic repeats (CRISPRs) and five of six cas (CRISPR-associated) genes. Our data suggest a complex interaction between microbe and bacteriophage impacts the group behaviors of P. aeruginosa. MATERIALS AND METHODSBacterial and phage culture preparation. Strains and plasmids used in this study are shown in Table 1. Overnight cultures were streaked from glycerol stocks stored at Ϫ80°C onto lysogeny broth (LB) agar (1.5%) and incubated overnight at 37°C to isolated single colonies. These colonies were then used to inoculate plankt...
In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.
Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.
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