Type IV pili of Enterobacteriaceae species.

Type IV pili (T4Ps) are surface filaments widely distributed among bacteria and archaea. T4Ps are involved in many cellular functions and contribute to virulence in some species of bacteria. Due to the diversity of T4Ps, different properties have been observed for homologous proteins that make up T4Ps in various organisms. In this review, we highlight the essential components of T4Ps, their functions, and similarities to related systems. We emphasize the unique T4Ps of enteric pathogens within the Enterobacteriaceae family, which includes pathogenic strains of Escherichia coli and Salmonella. These include the bundle-forming pilus (BFP) of enteropathogenic E. coli (EPEC), longus (Lng) and colonization factor III (CFA/III) of enterotoxigenic E. coli (ETEC), T4P of Salmonella enterica serovar Typhi, Colonization Factor Citrobacter (CFC) of Citrobacter rodentium, T4P of Yersinia pseudotuberculosis, a ubiquitous T4P that was characterized in enterohemorrhagic E. coli (EHEC), and the R64 plasmid thin pilus. Finally, we highlight areas for further study.

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

A non-pediocin low molecular weight antimicrobial peptide produced by Pediococcus pentosaceus strain IE-3 shows increased activity under reducing environment.

BACKGROUND: Species of the genus Pediococcus are known to produce antimicrobial peptides such as pediocin-like bacteriocins that contain YGNGVXC as a conserved motif at their N-terminus. Until now, the molecular weight of various bacteriocins produced by different strains of the genus Pediococcus have been found to vary between 2.7 to 4.6 kD. In the present study, we characterized an antimicrobial peptide produced by P. pentosaceus strain IE-3. RESULTS: Antimicrobial peptide was isolated and purified from the supernatant of P. pentosaceus strain IE-3 grown for 48 h using cation exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) techniques. While MALDI-TOF MS experiments determined the precise molecular mass of the peptide to be 1701.00 Da, the de novo sequence (APVPFSCTRGCLTHLV) of the peptide revealed no similarity with reported pediocins and did not contain the YGNGVXC conserved motif. Unlike pediocin-like bacteriocins, the low molecular weight peptide (LMW) showed resistance to different proteases. Moreover, peptide treated with reducing agent like dithiothreitol (DTT) exhibited increased activity against both Gram-positive and Gram-negative test strains in comparison to native peptide. However, peptide treated with oxidizing agent such as hydrogen peroxide (H2O2) did not show any antimicrobial activity. CONCLUSION: To our knowledge this is the lowest molecular weight peptide produced by members of the genus Pediococcus. The low molecular weight peptide shared amino acid arrangement with N-terminal sequence of Class IIa, pediocin-like bacteriocins and showed increased activity under reducing conditions. Antimicrobial peptides active under reduced conditions are valuable for the preservation of processed foods like meat, dairy and canned foods where low redox potential prevails.

Genome sequence of Brevibacillus laterosporus strain GI-9.

We report the 5.18-Mb genome sequence of Brevibacillus laterosporus strain GI-9, isolated from a subsurface soil sample during a screen for novel strains producing antimicrobial compounds. The draft genome of this strain will aid in biotechnological exploitation and comparative genomics of Brevibacillus laterosporus strains.

Phosphorylation of PhoP protein plays direct regulatory role in lipid biosynthesis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis PhoP is essential for virulence and intracellular growth of the tubercle bacilli. Genetic evidence suggests that PhoP regulates complex lipid biosynthesis, and absence of some of these lipid molecules in a phoP mutant partly accounts for its attenuated growth in macrophages and/or mice. To investigate the mechanism of regulation, here we demonstrate the essentiality of phosphorylation of PhoP in the regulation of complex lipid biosynthesis. We show that phosphorylated PhoP activates transcription of pks2 and msl3, gene(s) encoding polyketide ß-ketoacyl synthases through direct DNA binding at the upstream regulatory region(s) of the target genes. Our results identify the genetic determinants recognized by PhoP and show that activation of target genes requires interaction(s) of the phosphorylated regulator at the cognate binding sites. The fact that these sites within the regulatory region of respective genes do not bind in vitro with either unphosphorylated or phosphorylation-deficient PhoP protein is consistent with phosphorylation-dependent assembly of the transcription initiation complex leading to in vivo transcriptional activation. Together, these results reveal so far unknown molecular mechanisms of how PhoP contributes to M. tuberculosis cell wall composition by regulating complex lipid biosynthesis.

Micrococcus lactis sp. nov., isolated from dairy industry waste.

A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).