A prototrophic suppressor of a Vibrio fischeri D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >1011 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative broad-spectrum racemase of V. fischeri, bsrF. An engineered bsrF allele lacking the putative secretion signal (ΔSS-bsrF) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS-bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS-bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance. IMPORTANCE: D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.

Transient infection of Euprymna scolopes with an engineered D-alanine auxotroph of Vibrio fischeri.

The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase (alr) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an ∆alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC, which encodes cystathionine ß-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the ∆alr mutation with deletions of metC and/or bsrF, which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on ∆alr ∆bsrF mutant MC13, which has a suppression rate of <10-9. When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ.IMPORTANCEThe importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between Vibrio fischeri and Euprymna scolopes. First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that metC obviates the need for D-ala supplementation of an alr mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in Escherichia coli regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid-Vibrio symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of V. fischeri within its host.

Evidence for Layer-Specific Connectional Heterogeneity in the Mouse Auditory Corticocollicular System.

The auditory cortex (AC) sends long-range projections to virtually all subcortical auditory structures. One of the largest and most complex of these-the projection between AC and inferior colliculus (IC; the corticocollicular pathway)-originates from layer 5 and deep layer 6. Though previous work has shown that these two corticocollicular projection systems have different physiological properties and network connectivities, their functional organization is poorly understood. Here, using a combination of traditional and viral tracers combined with in vivo imaging in both sexes of the mouse, we observed that layer 5 and layer 6 corticocollicular neurons differ in their areas of origin and termination patterns. Layer 5 corticocollicular neurons are concentrated in primary AC, while layer 6 corticocollicular neurons emanate from broad auditory and limbic areas in the temporal cortex. In addition, layer 5 sends dense projections of both small and large (>1 µm2 area) terminals to all regions of nonlemniscal IC, while layer 6 sends small terminals to the most superficial 50-100 µm of the IC. These findings suggest that layer 5 and 6 corticocollicular projections are optimized to play distinct roles in corticofugal modulation. Layer 5 neurons provide strong, rapid, and unimodal feedback to the nonlemniscal IC, while layer 6 neurons provide heteromodal and limbic modulation diffusely to the nonlemniscal IC. Such organizational diversity in the corticocollicular pathway may help to explain the heterogeneous effects of corticocollicular manipulations and, given similar diversity in corticothalamic pathways, may be a general principle in top-down modulation.SIGNIFICANCE STATEMENT We demonstrate that a major descending system in the brain is actually two systems. That is, the auditory corticocollicular projection, which exerts considerable influence over the midbrain, comprises two projections: one from layer 5 and the other from layer 6. The layer 6 projection is diffusely organized, receives multisensory inputs, and ends in small terminals; while the layer 5 projection is derived from a circumscribed auditory cortical area and ends in large terminals. These data suggest that the varied effects of cortical manipulations on the midbrain may be related to effects on two disparate systems. These findings have broader implications because other descending systems derive from two layers. Therefore, a duplex organization may be a common motif in descending control.