The bacterial cell wall is a highly conserved essential component of most bacterial groups. It is the target for our most frequently used antibiotics and provides important small molecules that trigger powerful innate immune responses. The wall is composed of glycan strands crosslinked by short peptides. For many years, the penicillin-binding proteins were thought to be the key enzymes required for wall synthesis. RodA and possibly other proteins in the wider SEDS (shape, elongation, division and sporulation) family have now emerged as a previously unknown class of essential glycosyltranferase enzymes, which play key morphogenetic roles in bacterial cell wall synthesis. We provide evidence in support of this role and the discovery of small natural product molecules that probably target these enzymes. The SEDS proteins have exceptional potential as targets for new antibacterial therapeutic agents.

Streptomycetes sense and respond to the stress of phosphate starvation via the two-component PhoR–PhoP signal transduction system. To identify the in vivo targets of PhoP we have undertaken a chromatin-immunoprecipitation-on-microarray analysis of wild-type and phoP mutant cultures and, in parallel, have quantified their transcriptomes. Most (ca. 80%) of the previously in vitro characterized PhoP targets were identified in this study among several hundred other putative novel PhoP targets. In addition to activating genes for phosphate scavenging systems PhoP was shown to target two gene clusters for cell wall/extracellular polymer biosynthesis. Furthermore PhoP was found to repress an unprecedented range of pathways upon entering phosphate limitation including nitrogen assimilation, oxidative phosphorylation, nucleotide biosynthesis and glycogen catabolism. Moreover, PhoP was shown to target many key genes involved in antibiotic production and morphological differentiation, including afsS, atrA, bldA, bldC, bldD, bldK, bldM, cdaR, cdgA, cdgB and scbR-scbA. Intriguingly, in the PhoP-dependent cpk polyketide gene cluster, PhoP accumulates substantially at three specific sites within the giant polyketide synthase-encoding genes. This study suggests that, following phosphate limitation, Streptomyces coelicolor PhoP functions as a ‘master’ regulator, suppressing central metabolism, secondary metabolism and developmental pathways until sufficient phosphate is salvaged to support further growth and, ultimately, morphological development.

Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P i ) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2,3 cyclic nucleotide 2-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3 or 5 phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.When Bacillus subtilis encounters phosphate starvation stress, it responds by inducing groups of genes that function to restrict the metabolic consequences of the limited supply of this essential nutrient. These groups of genes are collectively referred to as the phosphate (Pho) stimulon. The phosphate stimulon includes at least two well-described regulons, namely, the sigma B ( B ) general stress regulon and the phosphate starvation-specific PhoP regulon. When B. subtilis encounters phosphate starvation, genes of the SigB regulon are induced by the alternative sigma factor, B , and genes of the PhoP regulon are either induced or repressed by activated PhoP (namely, PhoPϳP). The B general stress regulon contains Ͼ100 genes (58, 64). These genes provide a nonspecific response to stress by encoding proteins that protect the DNA, membranes, and proteins from the damaging effects of stress. Proteins induced by B help the cell to survive potentially harmful environmental conditions, such as heat, osmotic, acid, or alkaline shock (6,21,23,26). This protective function is thought to be particularly important in maintaining the viability of nongrowing cells.The PhoP regulon currently consists of 34 members. Six operons (phoPR [56,60], phoB-ydhF [7,14], pstSAC-pstBApstBB [3,67], phoD-tatAD [7,19], resABCDE [10], and tuaABC DEFGH [40,72]) and five monocistronic genes (glpQ [7], phoA [30,31], tatCD [34], ykoL [60], and yttP [62]) are induced and two operons (tagAB and tagDEF (39) are repressed in response to phosphate starvation. phoA and phoB encode alkaline phosphatases (APases) which facilitate the recovery of inorganic phosphate (P i ) from organic sources (11, 30); phoD encodes a phosphodiesterase/APase, putatively involved in cell wall teichoic acid turnover, and is secreted exclus...

When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation. The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA. The phoPR operon is transcribed from two A -dependent promoters, P 1 and P 2 . Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR-null mutant), a low level of phoPR transcription was detected only from promoter P 1 . During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoPϳP)-dependent manner; in addition to P 1 , the P 2 promoter becomes active. In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoPϳP could bind to the control region of the phoPR operon. The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoPϳP to induce other members of the Pho regulon. The extent to which promoters P 1 and P 2 are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition. For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH, sigF, and sigE mutants. The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.

SummaryAntibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered—compared with rifampicin—conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.