Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient in inducible nitric oxide synthase did not support the growth of E. coli by nitrate respiration, suggesting that the nitrate generated during inflammation was host-derived. Thus, the inflammatory host response selectively enhances the growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.

https://science.sciencemag.org/content/sci/339/6120/708.full.pdf

Host-Derived Nitrate Boosts Growth of E. coli in the Inflamed Gut

Supplementary Materials for Host-Derived Nitrate Boosts Growth of E. coli in the Inflamed Gut

Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues. Quantitative, unbiased study of these spatio-temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high-throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini-colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old-pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position-dependent silencing is regulated by division.

High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.

Inflammatory diseases of the gastrointestinal tract are frequently associated with dysbiosis, characterized by changes in gut microbial communities that include an expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Here we show that a dysbiotic expansion of Enterobacteriaceae during gut inflammation could be prevented by tungstate treatment, which selectively inhibited molybdenum-cofactor-dependent microbial respiratory pathways that are operational only during episodes of inflammation. By contrast, we found that tungstate treatment caused minimal changes in the microbiota composition under homeostatic conditions. Notably, tungstate-mediated microbiota editing reduced the severity of intestinal inflammation in mouse models of colitis. We conclude that precision editing of the microbiota composition by tungstate treatment ameliorates the adverse effects of dysbiosis in the inflamed gut.

/pdf/precision-editing-of-the-gut-microbiota-ameliorates-colitis-ouqqm1qng7.pdf

Precision editing of the gut microbiota ameliorates colitis

The bacterial MreB actin cytoskeleton is required for cell shape maintenance in most non-spherical organisms. In rod-shaped cells such as Escherichia coli, it typically assembles along the long axis in a spiral-like configuration just underneath the cytoplasmic membrane. How this configuration is controlled and how it helps dictate cell shape is unclear. In a new genetic screen for cell shape mutants, we identified RodZ (YfgA) as an important transmembrane component of the cytoskeleton. Loss of RodZ leads to misassembly of MreB into non-spiral structures, and a consequent loss of cell shape. A juxta-membrane domain of RodZ is essential to maintain rod shape, whereas other domains on either side of the membrane have critical, but partially redundant, functions. Though one of these domains resembles a DNA-binding motif, our evidence indicates that it is primarily responsible for association of RodZ with the cytoskeleton.

/pdf/rodz-yfga-is-required-for-proper-assembly-of-the-mreb-actin-2sgv87e61z.pdf

RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli.

Replication initiator proteins in bacteria not only allow DNA replication but also often regulate the rate of replication initiation as well. The regulation is mediated by limiting the synthesis or availability of initiator proteins. The applicability of this principle is demonstrated here for RctB, the replication initiator for the smaller of the two chromosomes of Vibrio cholerae. A strong promoter for the rctB gene named rctBp was identified and found to be autoregulated in Escherichia coli. Promoter activity was lower in V. cholerae than in E. coli, and a part of this reduction is likely to be due to autorepression. Sequences upstream of rctBp, implicated earlier in replication control, enhanced the repression. The action of the upstream sequences required that they be present in cis, implying long-range interactions in the control of the promoter activity. A second gene specific for chromosome II replication, rctA, reduced rctB translation, most likely by antisense RNA control. Finally, optimal rctBp activity was found to be dependent on Dam. Increasing RctB in trans increased the copy number of a miniplasmid carrying oriCIIVC, implying that RctB can be rate limiting for chromosome II replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis.

Multipartite Regulation of rctB, the Replication Initiator Gene of Vibrio cholerae Chromosome II

Most bacteria have one chromosome but some have more than one, as is common in eukaryotes. How multiple chromosomes are maintained in bacteria remains largely obscure. Here we have examined the behaviour of the two Vibrio cholerae chromosomes as a function of growth rate. At slow growth rates, both chromosomes were maintained at copy numbers of one to two per cell. Increasing the growth rate by nutritional shift-up amplified the origin-proximal DNA of the larger chromosome (chrI) to four copies per cell, but not that of the smaller chrII. The latter was amplified when its specific initiator was supplied in excess or a specific negative regulator was deleted. The growth rate-insensitive behaviour of chrII, whose origin is similar to origins of members of a major class of plasmids, was shared by some but not all of several representative plasmids tested in V. cholerae. Also, unlike plasmid replication, chrII replication is known to be initiated at a specific stage of the cell cycle. Raising chrII copy number decreased growth rate, suggesting that this chromosome might serve as a repository for necessary but potentially deleterious genes.

Selective chromosome amplification in Vibrio cholerae

MreB is an actin homolog required for the morphogenesis of most rod-shaped bacteria and for other functions, including chromosome segregation. In Caulobacter crescentus and Escherichia coli, the protein seems to play a role in the segregation of sister origins, but its role in Bacillus subtilis chromosome segregation is less clear. To help clarify its role in segregation, we have here studied the protein in Vibrio cholerae, whose chromosome I segregates like the one in C. crescentus and whose chromosome II like the one in E. coli or B. subtilis. The properties of Vibrio MreB were similar to those of its homologs in other bacteria in that it formed dynamic helical filaments, was essential for viability, and was inhibited by the drug A22. Wild-type (WT) cells exposed to A22 became spherical and larger. The nucleoids enlarged correspondingly, and the origin positions for both the chromosomes no longer followed any fixed pattern. However, the sister origins separated, unlike the situation in other bacteria. In mutants isolated as A22 resistant, the nucleoids in some cases appeared compacted even when the cell shape was nearly normal. In these cells, the origins of chromosome I were at the distal edges of the nucleoid but not all the way to the poles where they normally reside. The sister origins of chromosome II also separated less. Thus, it appears that the inhibition or alteration of Vibrio MreB can affect both the nucleoid morphology and origin localization.

Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae

Genome duplication and segregation normally are completed before cell division in all organisms. The temporal relation of duplication and segregation, however, can vary in bacteria. Chromosomal regions can segregate towards opposite poles as they are replicated or can stay cohered for a considerable period before segregation. The bacterium Vibrio cholerae has two differently sized circular chromosomes, chromosome I (chrI) and chrII, of about 3 and 1 Mbp, respectively. The two chromosomes initiate replication synchronously, and the shorter chrII is expected to complete replication earlier than the longer chrI. A question arises as to whether the segregation of chrII also is completed before that of chrI. We fluorescently labeled the terminus regions of chrI and chrII and followed their movements during the bacterial cell cycle. The chrI terminus behaved similarly to that of the Escherichia coli chromosome in that it segregated at the very end of the cell division cycle: cells showed a single fluorescent focus even when the division septum was nearly complete. In contrast, the single focus representing the chrII terminus could divide at the midcell position well before cell septation was conspicuous. There were also cells where the single focus for chrII lingered at midcell until the end of a division cycle, like the terminus of chrI. The single focus in these cells overlapped with the terminus focus for chrI in all cases. It appears that there could be coordination between the two chromosomes through the replication and/or segregation of the terminus region to ensure their segregation to daughter cells.

Segregation of the replication terminus of the two Vibrio cholerae chromosomes.

The bacterium Vibrio cholerae has two chromosomes. The origin of replication of chromosome I is similar to that of Escherichia coli. The origin-containing region of chromosome II (oriCII) resembles replicons of plasmids such as P1, except for the presence of an additional gene, rctA [Egan, E. S. & Waldor, M. K. (2003) Cell 114, 521–530]. The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.

https://www.pnas.org/content/pnas/103/32/12051.full.pdf

Preeti Srivastava

Papers

Multipartite Regulation of rctB, the Replication Initiator Gene of Vibrio cholerae Chromosome II

Selective chromosome amplification in Vibrio cholerae

Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae

Segregation of the replication terminus of the two Vibrio cholerae chromosomes.

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II.